The neurotoxic effects of bilirubin may involve modulation of neuronal protein phosphorylation systems. Using in vitro phosphorylation assays and a variety of protein substrates and purified protein kinases, we have studied the mechanism of bilirubin-induced inhibition of protein phosphorylation. Bilirubin was found to inhibit cAMP-dependent, cGMP-dependent, Ca(2+)- calmodulin-dependent, and Ca(2+)-phospholipid-dependent protein kinases, irrespective of substrate properties. Fifty percent inhibition occurred at bilirubin concentrations varying from 20 to 125 microM. Kinetic analysis, using the isolated catalytic subunit of cAMP-dependent kinase and a synthetic peptide substrate derived from the protein phospholemman, indicated that bilirubin (50 microM) decreased the apparent Vmax of the reaction, irrespective of whether ATP or peptide levels were varied, without significantly altering the apparent K(m) value. Thus our results indicate that bilirubin can inhibit catalytic domain(s) of protein kinases by apparent noncompetitive mechanism(s), presumably by interacting with noncatalytic domains on the enzyme. Given the key role of protein phosphorylation in cellular regulation, the widespread inhibitory effect of bilirubin on protein kinases may contribute to bilirubin neurotoxicity.