The intracellular membranes of hepatomas exhibit an altered content and composition of lipid compared to the membranes of normal liver. In order to elucidate the role of lipid biosynthetic enzymes in these membrane differences, we first examined the fatty acyl-CoA ligase and acyl-CoA:sn-glycerol 3-phosphate (Gro-3P) acyltransferase activities and acyl specificities of microsomes from liver, Morris hepatoma 7288C, and hepatoma tissue culture (HTC) cells. Based upon incorporation of fatty acid and Gro-3P, it is concluded that acyl-CoA:sn-Gro-3P acyltransferase activities are markedly elevated (6-30-fold) in the microsomes of Morris Hepatoma 7288C and HTC cells compared to microsomes from liver, whereas the fatty acyl-CoA ligase activity is reduced (30-50-fold). Therefore, the low phospholipid content of these tumor cells does not appear to result from reduced acyltransferase activity. Though diminished ligase activity may play a role, it appears that activation of fatty acid may not be rate-limiting, even at the low levels of fatty acyl-CoA ligase present in the tumor and HTC cells. Preliminary evidence suggests that another factor that may be responsible for the low tumor phospholipid content is the limited availability of Gro-3P, a lipid precursor. The phospholipid in hepatoma 7288C is also characterized by an elevated ratio of monenoic to dienoic fatty acid. We have found that this change does not reflect an altered specificity of acyl-CoA:sn-Gro-3P acyltransferase.