The proximate cholesterol precursors lathosterol, 7-dehydrocholesterol and desmosterol supported the growth of NS-1 and X63 mouse myeloma cells. These cells and X63.653 cells are cholesterol auxotrophs, yet each was able to convert [3H]lathosterol to [3H]cholesterol. These results are consistent with the conclusion that cholesterol auxotrophy in these myeloma cells is due to a deficiency in 3-ketosteroid reductase activity. The steroid hormones testosterone, progesterone and hydrocortisone could not replace cholesterol as a medium supplement. These results provide a greater understanding of the cholesterol auxotrophy characteristic of cell lines clonally-derived from the MOPC 21 myeloma tumor, and they provide a rational basis for the use of sterols in defined culture medium for mouse myeloma cells.