The relationship between age-related alterations in the lipid composition of cultured rat-heart fibroblasts and several biochemical and biophysical parameters was investigated. Aged (14-15-day-old) cultures displayed higher mole ratios of sphingomyelin to phosphatidylcholine, as well as elevated cholesterol levels. A concomitant increase was observed in the total protein content of the cells and in the Vmax values of both membranal and cytoplasmic marker enzymes. Fluorescence photobleaching recovery was employed to study the lateral mobility of the lipid probe NBD-phosphatidylethanolamine and of membrane glycoproteins that bind succinylated concanavalin A. The mobile fractions of both probes were higher in aged cultures, while the lateral diffusion coefficients were lower. To further demonstrate the dependence of the above parameters on the cellular lipid composition, we have manipulated the lipid composition of old cultures by treatments with liposomes (small unilamellar vesicles) of specific compositions. Treatments which reversed the lipid composition towards that of young (5-6-day-old) cultures caused a concomitant reversal of the measured biochemical and biophysical parameters to the values observed in young cultures. These findings suggest that alterations in the organization and mobility of cell membrane constituents are involved in mediating changes in cellular functions. In view of our previous findings on cultures of rat-heart myocytes (Yechiel, E., Barenholz, Y. and Henis, Y.I. (1985) J. Biol. Chem. 260, 9132-9136), it appears that the modulation of cellular properties through the membrane lipid composition may be a general phenomenon in many cell types.