The concentration of different prostaglandins (PG's) was determined in both cells and culture medium of growing BALB/3T3 and BALB/3T3 (3T3) cultures transformed by simian virus 40 (SV3T3). Most PG's were found in the culture medium rather than in the cells. Further, the larger PG measurements were PGE and a composite measurement of PGA and PGB. PGF was detected at lower levels. The sum of PGE and the composite measurement (PGA+PGB) was the best indication of PG production in culture. When 2-day medium collections from 3T3 and SV3T3 cells were measured by radioimmunoassay for PGs, higher concentrations of PG were detected in the media of SV3T3 cultures. This difference in PG production was not due merely to differences in cell density, since SV3T3 cells produced higher PG concentrations, even at equal cell densities. PG production for a 2-day interval was more a function of cell type than cell density.