The studies presented herein were designed to investigate the role of insulin in maintaining the steady-state number of ribosomes in primary cultures of rat hepatocytes. The RNA content of hepatocytes maintained in the presence of insulin did not change during the 7 days of culture. In contrast, the RNA content declined significantly following 2 days of insulin deprivation and after 4 days without insulin reached a new steady-state value that was approximately 60% of that observed for hepatocytes maintained in the presence of the hormone. Following addition of insulin, the RNA content of insulin-deprived hepatocytes started to increase within 6 h and was restored to the control value within 48 h. The amounts of 18 and 28 S RNA, and thus the number of ribosomes, changed in concert with the total RNA content. Furthermore, synthesis of total cellular protein responded in parallel to the changes in RNA content, suggesting that the number of ribosomes was the primary determinant of protein synthesis under the conditions of these experiments. About one-half of the increase in RNA content following the addition of insulin to insulin-deprived cells could be accounted for by a reduction in the rate of degradation of ribosomes. The remainder of the change in RNA content must have resulted from an increase in ribosome biogenesis. This possibility was confirmed by measuring [3H]uridine incorporation into ribosomal RNA present in mature ribosomes. The rate of synthesis of ribosomal RNA was reduced in parallel with the fall in RNA content in insulin-deprived hepatocytes and was restored to the control value within 3 h of the addition of insulin. Alterations in the synthesis of ribosomal RNA in response to insulin deprivation and replacement were associated with parallel changes in transcription of rDNA as measured in nuclear run-on assays using a DNA probe corresponding to the external transcribed spacer region of rat rDNA. The data demonstrate that insulin regulates the number of ribosomes in primary cultures of rat hepatocytes by accelerating the rate of transcription of rDNA and by slowing the rate of ribosome degradation.