The activity of many membrane proteins, such as receptors, ionic channels, transporters, and enzymes, is cholesterol dependent; however, mechanisms of the cholesteroldependent regulation of protein functions remain obscure. Recent studies suggest that membrane proteins can directly interact with cholesterol owing to the presence of the cholesterolrecognizing aminoacid consensus (CRAC) motifs. One of the ways to verify and further develop this notion is a design of CRACcontaining peptides and investigation of their effects on cholesteroldependent cell functions. Previously we showed that a newly constructed peptide RTKL ÏÏEMJLVJELGNM DKAVKLWRKLKR (peptide P4) containing two CRAC motifs modulates cholesteroldependent interactions of cultured macrophages IC21 with 2u.m particles. In this work, in order to clarify the role of CRACforming amino acids, we employed the same experimental system to test the activity of peptides closely related to P4 but with modified CRAC motifs. We found that peptide STKLSEMLSEL GNMDKASKL SRKLS R (Mut2) analogous to P4, except that all CRACforming amino acids were substituted by serine (V»S,W»S,R>S), did not produce any effect in the concentration range 0.550 fiM corresponding to the range of the P4 activity. Neither was effective peptide RTKLSEMLVEL GNMDKAVKLSRKLKR (Mut3), in which only aromatic amino acids of the C R A C motifs were substituted (W » S). Peptide STKLWEMLVEL GNMDKAVKLWRKLSR (Mut4), in which only cationic amino acids in the CRAC motifs were changed (R / K » S), produced almost the same effect as that of peptide P4 with a bellshape dose—response curve. At low concentrations (1 —4 ]iM) Mut4 notably increased the number of beads per cell, at higher concentrations this parameter diminished, and at 50 Ц.М Mut4 produced a robust toxic effect. Finally, peptide EWGMAYLffiEßNRKLKKDLKVLKMLRT (Muti) composed of the same amino acid residues as P4 but in a random order ("scramble") and possessing one CRAC motif, different from that in P4, produced a moderate stimulation at 4—10 |J.M but was not toxic at 50 u.M. As in the case of peptide P4, the effects of Mut4 and Muti depended on the cholesterol content in the cell membrane: after the incubation of cells with cholesterolextracting agent methylßcyclodextrin stimulatory effects produced by Mut4 and Muti at low doses were suppressed. Our results indicate that CRAC motifs play an important role in the mechanisms of the peptide-induced modulations of cholesterol-dependent cell functions in the experimental system used and that of the three motif-forming amino acids, critical is the presence of aromatic amino acids (W). Further research is required to comprehend the molecular mechanisms of interactions of CRAC-containing peptides with cell membrane components that lead to modulation of cell functions. We anticipate that CRAC-containing peptides may provide a basis for the development of new tools for directed regulation of the activity of target cholesterol-dependent membrane proteins and for the design of new antimicrobial and immunomodulating drugs in particular.