We have shown that NS-1 mouse myeloma cells, but not NS-1 hybridomas, required human low density lipoprotein (LDL) for survival and growth in serum-free cell culture (Kawamoto et al., 1983). Here we have further defined the lipid requirement of NS-1 cells by demonstrating that LDL could be replaced by cholesterol complexed with carrier bovine serum albumin (BSA). Cholesterol was the active component of this complex since BSA alone did not promote NS-1 survival or growth. Cholesterol was an absolute requirement of these cells, and it could not be replaced by mevalonolactone. In contrast to NS-1 cells a related mouse myeloma cell line, Sp2/0, did not require cholesterol or other lipids for growth in vitro. Finally, we propose that cholesterol-deficient medium may be an effective alternative to HAT medium for selecting NS-1 hybridomas.