Simian virus 40 (SV40)-infected rat hepatocytes were tested for albumin production (a marker of differentiated hepatocyte function) by growth in serum-free medium for 30-40 days. We observed a biphasic peak of albumin production, with the first peak at 4-6 and the second at 10-20 days after plating. A 2- to 3-fold increase in the second peak was observed when albumin production by SV40-infected cultures was compared with that by uninfected cultures. No increase in cell number in SV40-infected cultures was observed during this 20-day period. By 25-35 days, albumin production by uninfected cultures ceased but was detected in 50-60% of SV40-infected cultures. SV40-induced stimulation and persistence occurred regardless of the attachment surface and did not require collagen. Transformation occurred regardless of whether stimulation or persistence of albumin production was seen; however, stimulation and persistence were necessary to yield transformed cultures that produced albumin.