The influence of diabetes and insulin treatment on the phospholipid content of R3230AC mammary tumors, a hormonally responsive neoplasm, was studied. Diabetes was induced by administration of streptozotocin 3 days prior to tumor implantation. Protamine zinc insulin, 3 IU/rat twice daily, was administered to tumor-bearing rats for 3 days. Enzymatically dissociated tumor cells from diabetic animals showed significant increases in phosphatidyl choline, lysophosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, and phosphatidic acid, compared to controls. Diabetic animals treated with insulin displayed reductions in phosphatidyl choline, lysophosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, and phosphatidic acid to levels approximating those found in intact (control) animals. However, neither diabetes nor insulin treatment altered sphingomyelin levels. Mammary tumor cells from diabetic animals showed a 21% increase in DNA content compared to that in intact controls and treatment of diabetic animals with insulin lowered DNA level significantly. The responsiveness of both phospholipids and DNA content to changes in the insulin milieu of the host suggest that phospholipids may play an important role in mediating the effects of insulin on growth of R3230AC tumors.