The effects of biotin upon the intracellular level of cGMP and the activity of glucokinase were examined in primary cultures of adult rat hepatocytes. The addition of biotin to the culture medium of hepatocytes increased their content of cGMP 3-fold within 1 h. A 4-fold increase in the activity of glucokinase was observed in response to the addition of the vitamin to the culture medium and the maximal response was observed 6 h following the addition to the medium. These maximum effects were noted when biotin was present in the culture medium at a concentration of 10(-6) M. The induction of glucokinase activity by biotin was preceded by an increase in the intracellular level of cGMP. The addition of 8-bromo-cGMP to the culture medium also increased the activity of glucokinase and its effects were not additive with respect to the effects of biotin. The induction of glucokinase by biotin or the cyclic nucleotide analog was not observed in the absence of insulin. The effects of biotin upon the activity of glucokinase could be mimicked by including glucose in the culture medium. When hexose utilization by the hepatocytes was blocked by the addition to the culture medium of N-acetylglucosamine, the induction of glucokinase by biotin was unaffected, whereas the induction brought about by glucose was not observed. The changes in the activity of the enzyme brought about by biotin or 8-bromo-cGMP was shown to arise as the result of changes in the rate of synthesis of the enzyme. In addition, using an in vitro translation assay and immunoprecipitation, it was found that biotin and 8-bromo-cGMP increased the amount of translatable mRNA coding for the enzyme.