Fluoride is a nucleophilic reagent which has been reported to inhibit a variety of different enzymes such as esterases, asymmetrical hydrolases and phosphatases. In this report, we demonstrate that fluoride inhibits tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle and human placenta. Fluoride inhibited in a similar dose-dependent manner both beta-subunit autophosphorylation and tyrosine kinase activity for exogenous substrates. This inhibitory effect of fluoride was not due to the formation of complexes with aluminum and took place in the absence of modifications of insulin-binding properties of the insulin receptor. Fluoride did not complete with the binding site for ATP or Mn2+. Fluoride also inhibited the autophosphorylation and tyrosine kinase activity of receptors for insulin-like growth factor I from human placenta. Addition of fluoride to the pre-phosphorylated insulin receptor produced a slow (time range of minutes) inhibition of receptor kinase activity. Furthermore, fluoride inhibited tyrosine kinase activity in the absence of changes in the phosphorylation of prephosphorylated insulin receptors, and the sensitivity to fluoride was similar to the sensitivity of the unphosphorylated insulin receptor. The effect of fluoride-on tyrosine kinase activity was markedly decreased when insulin receptors were preincubated with the copolymer of glutamate/tyrosine. Prior exposure of receptors to free tyrosine or phosphotyrosine also prevented the inhibitory effect of fluoride. However, the protective effect of tyrosine or phosphotyrosine was maximal at low concentrations, suggesting the interaction of these compounds with the receptor itself rather than with fluoride. These data suggest: (i) that fluoride interacts directly and slowly with the insulin receptor, which causes inhibition of its phosphotransferase activity; (ii) that the binding site of fluoride is not structurally modified by receptor phosphorylation; and (iii) based on the fact that fluoride inhibits phosphotransferase activity in the absence of alterations in the binding of ATP, Mn2+ or insulin, we speculate that fluoride binding might affect the transfer of phosphate from ATP to the tyrosine residues of the beta-subunit of the insulin receptor and to the tyrosine residues of exogenous substrates.