The available information about the specificity determinants of casein kinases-1 and -2 (CK1 and CK2) has been utilized to obtain two new peptide substrates optimally suited for the specific monitoring of these two pleiotropic enzymes. The best substrate developed for CK1 is the inhibitor-2 derived peptide RRKDLHDDEEDEAMSITA which is superior in every respect to all the non phosphorylated CK1 peptide substrates used so far. Its Km is 172 microM and the Vmax is 6-fold higher than that of casein. The dodecapeptide RRRADDSDDDDD, on the other hand, is totally refractory to CK1 while it is an excellent substrate for CK2, exhibiting, under basal conditions, a Km value of 19 microM and a Vmax higher than those obtained with all the routinely used substrates of CK2. Both the novel CK1 and CK2 peptide substrates are suited for the phosphocellulose paper assay.