The ribonucleoprotein ribonuclease P (RNase P) cleaves all tRNA precursors endonucleolitically to produce the mature 5'-end. Dictyostelium discoideum RNase P displays an absolute requirement for Mg2+. Only the alkaline earth cations Ca2+, Sr2+, and Ba2+, under appropriate conditions can substitute to some extent for Mg2+. The transition metals Mn2+, Co2+, Ni2+, and Cd2+ are efficient inhibitors of the enzyme activity. Ca2+, Sr2+ and Ba2+, in the presence of Mg2+, exhibit a bimodal action at the kinetic phase of the reaction. Kinetic analysis of the activation phase revealed that Ca2+, Sr2+, or Ba2+ attached on a specific site of RNase P act as nonessential-noncompetitive activators. Further additions of Ca2+, Sr2+, or Ba2+ cause noncompetitive inhibition on the RNase P reaction, indicating that RNase P possesses a second binding site responsible for the inhibitory effect of Ca2+, Sr2+, and Ba2+. Both activator and inhibitory sites can be occupied by Ca2+, Sr2+, or Ba2+ at the same time.