Long-term primary cultures of mouse mammary tumor cells proved an excellent source of mouse mammary tumor virus (MMTV). Virus purified from these primary cultures had the same morphologic, biochemical, immunologic, and biologic characteristics as MMTV. Quantitation of MMTV-protein equivalents released into the medium was measured by the radioimmunoassay for MMTV. Peak production levels were 20–40 µg MMTV protein equivalents/75-cm2 flask/24 hours. These cultures produced MMTV for as long as 90 days. MMTV cultivation depended on the initial cell-plating density and hormones. Maximal MMTV release was obtained at a plating density of 1×106 cells/cm2 in the presence of insulin and hydrocortisone. Insulin alone gave basal levels of MMTV, and hydrocortisone alone increased MMTV release only threefold, but insulin and hydrocortisone together effected an eightfold increase in MMTV release. This suggested that hydrocortisone had a primary effect on MMTV release and insulin acted synergistically with hydrocortisone to maximize MMTV release.