Damnacanthal, an anthraquinone isolated from a plant extract, was found to be a potent, selective inhibitor of p561(lck) tyrosine kinase activity. The structure, potency, and selectivity of damnacanthal were confirmed by independent synthesis and testing. Damnacanthal exhibited an IC50 of 17 nM for inhibition of p56(lck) autophosphorylation and an IC50 of 620 nM for phosphorylation of an exogenous peptide by p56(lck). Damnacanthal had > 100-fold selectivity for p56(lck) over the serine/threonine kinases, protein kinase A and protein kinase C, and >40-fold selectivity for p56(lck) over four receptor tyrosine kinases. It also demonstrated modest (7-20-fold), but highly statistically significant, selectivity for p56(lck) over the homologous enzymes p60(src) and p59(fyn). Mechanistic studies demonstrated that damnacanthal was competitive with the peptide binding site, but mixed noncompetitive with the ATP site. Although damnacanthal contains a potentially reactive aldehyde moiety, equilibrium dialysis experiments demonstrated that significant imine formation between damnacanthal and amines occurred only at high concentrations of reactants. However, damnacanthal appeared to bind nonspecifically to membrane lipids and was not active in whole cell tyrosine kinase assays. Damnacanthal is the most patent, selective inhibitor of p56(lck) tyrosine kinase activity described to date and may represent the starting point for the identification of novel, selective inhibitors of p56(lck) which are active in whole cell as well as in cell-free systems.