Specific cDNA probes were used to determine steady-state mRNA levels for the multiple glutathione S-transferases in primary hepatocyte cultures. In the first 24 hr of culture, gene transcripts for the Ya family decreased sharply, Yb3 disappeared completely, but changes in levels of mRNA for Yb1 and Yb2 were smaller. These results suggest that the isoenzymes are regulated independently. Yp mRNA, which is present at greatly elevated levels in hyperplastic nodules and hepatocellular carcinomas but not in normal adult livers, was hardly detectable in freshly isolated hepatocytes, but Yp transcripts rapidly accumulated in the first 24 hr in culture and continued to increase for 72 hr. Decreased levels in Ya and Yc and increases in Yp were detected by immunoblotting methods, indicating that translation products changed together with mRNA levels in the cultured cells. The appearance of Yp transcripts in hepatocytes was effectively blocked by addition of dexamethasone to the culture medium. Elevations of Yp levels are characteristic of the cell culture system and factors regulating Yp transcription in nodules and carcinomas may also be operative in cultured hepatocytes.