A series of 72 acridine and azaacridine derivatives have been tested as inhibitors of Ca(2+)- and phospholipid-activated protein kinase C (PKC), the catalytic subunit of cyclic AMP-dependent protein kinase (cAK) and Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK). A series of monomethyl derivatives of N-(2-dimethylaminoethyl)-acridine-4-carboxamide that have anti-tumour activity are good inhibitors of MLCK (IC50 values ranging from 4-138 microM) but these same compounds are ineffective or relatively poor inhibitors of PKC or cAK. With only several exceptions, the effective azaacridine inhibitors of PKC and MLCK (IC50 values < 200 microM) have basic or neutral 4-substituents whereas the effective azaacridine inhibitors of cAK have either neutral or acidic 4-substituents. The four exceptions found to this generality are effective inhibitors of all three protein kinases with IC50 values of about 100 microM or less. With several exceptions azaacridine inhibitors of PKC are also inhibitors of MLCK but effective inhibitors of cAK are relatively poor inhibitors of MLCK and PKC and vice versa. 4-N-(2-dimethylaminoethyl)-6-azaacridone-4-carboxamide is a competitive inhibitor of MLCK with respect to both the peptide substrate and ATP. All other acridines and azaacridines examined are non-competitive inhibitors of both MLCK and cAK with respect to both ATP and peptide substrate. All azaacridine PKC inhibitors examined are competitive with respect to ATP and noncompetitive with respect to peptide substrate suggesting that binding of these inhibitors is at or near the enzyme active site. The inhibition of MLCK (but not cAK or PKC) by anti-tumour acridine carboxamides suggests a novel site of in vivo biological action and a useful criterion for the detection of potential antitumour compounds.