Mg2+ as well as Mn2+, and Co2+, which may substitute Mg2+ in the mental ion requirement of casein kinase 2 (Gatica et al., FEBS Lett: 315:173-173, 1993), have been repeatedly reported to display an optimal concentration at which activity of casein kinase 2 is maximal. As far as we know this intriguing property has always been observed with casein as substrate. This phosphoprotein is not the natural substrate of the enzyme, and it is well known that it binds divalent metal ions, which provoke the aggregation and precipitation of the protein. Since an optimal concentration of metal ion might have a regulatory role, we have examined if it is a consequence of the particular properties of casein, or it is an inherent property of the enzyme, extensive to other substrates. We have used the type II regulatory subunit of protein kinase A which is a physiological substrate of the enzyme, and the peptide RRREEETEEE as a specific substrate. No optimal concentration of Mg2+ is observed when these two substrates are used. The results explain, however, why that optimum is observed with casein. Although low concentration of Mn2+, and Co2+ render about 25% of the maximal activity found with Mg2+, they inactivate the enzyme almost fully at concentrations at which Mg2+ yield the maximal activity.