In the present study the relationship between changes in tyrosine aminotransferase (TAT) enzyme activity, cytoplasmic mRNA levels, and gene transcription in response to both short- and long-term exposure to insulin was investigated. Insulin acutely inhibited transcription of the TAT gene by 50% in serum-deprived rat H4 hepatoma cells. Following this initial 50% decrease in transcription, there was a 2.5-fold induction in TAT activity that could not be accounted for by a concomitant increase in TAT mRNA levels. Insulin had no effect on the half-life of TAT mRNA. Insulin exposure for short periods of time also inhibited the glucocorticoid- and cAMP-induced transcription of the TAT gene. Like insulin, protein synthesis inhibitors acutely inhibited basal and glucocorti-coid-induced TAT transcription. TAT activity gradually returned toward basal levels after 8 h of insulin treatment. A second insulin-induced increase in TAT activity (3.5-fold above basal levels) was observed by 24 h of insulin treatment. This second phase of insulin-induced TAT activity was associated with elevated levels of TAT transcription and TAT mRNA levels, and, therefore, unlike the earlier stimulation, could be accounted for by changes in gene expression. Thus, the insulin-mediated regulation of the TAT gene in H4 cells is complex. Different transcriptional and post-transcriptional mechanisms are likely to be involved in the biphasic responses to insulin. © 1992.