The purpose of this work was to investigate whether biotin, a water-soluble vitamin, regulates the glucokinase gene. Biotin was administered intraperitoneally to starved rats, and the time course of glucokinase induction was followed over a time period of 12 h. The glucokinase mRNA was increased 19.6-fold during the first 1 h after biotin administration, afterwards rapidly decayed, and was hardly detectable by 4 h. The amount of glucokinase activity as determined by conventional enzyme activity assay increased in a time-dependent fashion, reaching 4-fold by 2 h of biotin administration. The transcriptional activity of the gene as measured by a nuclear run-on assay was increased about 6.7-fold within 45 min of biotin administration. These findings indicate that biotin can regulate the glucokinase gene at the transcriptional stage in the starved rat.