Mg(2+)-chelatase catalyses the first step unique to chlorophyll synthesis, namely the insertion of Mg2+ into protoporphyrin IX. When pea (Pisum sativum L., cv. Spring) chloroplasts are lysed in a buffer lacking Mg2+ and the thylakoids removed by centrifugation, the remaining mixture of light membranes and soluble proteins (LM/S) has high Mg(2+)-chelatase activity. Several lines of evidence are presented to show that the Mg2+ insertion catalysed by this preparation is a two-step reaction consisting of activation followed by Mg2+ chelation. An activated state of Mg(2+)-chelatase is achieved by preincubating LM/S with ATP. The activated state is observed as the elimination of the approx. 6 min lag in the rate of Mg2+ chelation on addition of the porphyrin substrate. The activity of LM/S assayed at low protein concentrations can be greatly enhanced by preincubating at high protein concentrations (12 mg/ml is optimal). This activation effect requires the presence of both LM and S fractions, as well as ATP. Both steps require ATP, but at different concentrations; the first step is optimal at > 0.5 mM (EC50 = 0.3 mM) and the second step is optimal at 0.3 mM (EC50 < 0.2 mM). ATP in the first step could be replaced by ATP[S]; this analogue could not sustain activity in the second step. This activated state was stable for at least 30 min at room temperature, but chilling of preincubated LM/S on ice for 30 min caused an almost complete loss of the activated state.