Sterols are universal constituents of eukaryotic membranes. Their abundance and intracellular disposition are under elaborate control, but we do not understand the central mechanisms underlying this regulation. Among the few known candidates for such functions are the OSBP, oxysterol binding proteins. Oxysterols serve as signal molecules in sterol homeostasis, and OH (OSBP homolog) proteins could comprise an ancient family which plays diverse roles in membrane lipid management. Because their mechanisms of action are not well understood, the OH family in the amoeba, Dictyostelium discoideum (the dOH) is being investigated. cDNA fragments for the five known dOH genes are being cloned and sequenced. These sequences will be analyzed in comparison with their diverse eukaryotic homologs. While the possibility of multiple functions for the dOH family is of great interest, those proteins which actually bind oxysterols and/or participate in sterol homeostasis are of greatest current interest. One clue to the function of the dOH will be their subcellular distribution and their possible redistribution in response to physiological and pharmacological effectors. The dispersion along the endocytic pathway of relevant dOH species will be of particular interest. Their mechanism of action will be pursued by correlating defined mutations with biochemical phenotypes. This inquiry will include defining their binding sites for oxysterols, membranes, self-association and association with other proteins. Chimeric constructs will help us to understand the specificity and commonality of the dOH domains.