The activation of human C1, a Ca(2+)-dependent complex proteinase comprising a non-enzymic protein, C1q, and two serine proteinases, C1r and C1s, is based primarily on the intrinsic property of C1r to autoactivate. The aim of the present study was to investigate the mechanisms involved in the regulation of C1r autoactivation, with particular attention to the role of Ca2+ ions. Spontaneous activation of proenzyme C1r was observed upon incubation in the presence of EDTA, whereas Ca2+ ions reduced markedly the activation process. Several lines of evidence indicated that Ca2+ inhibited the intramolecular activation reaction but had little or no effect on the intermolecular activation reaction. C1q caused partial release of this inhibitory effect of Ca2+. Complete stabilization of C1r in its proenzyme form was obtained upon incorporation within the Ca(2+)-dependent C1s-C1r-C1r-C1s tetramer, and a comparable effect was observed when C1s was replaced by its Ca(2+)-binding alpha-fragment. Both tetramers, C1s-C1r-C1r-C1s and C1s alpha-C1r-C1r-C1s alpha, readily associated with C1q to form 16.0 S and 14.7 S complexes respectively in which C1r fully recovered its activation potential. Both complexes showed indistinguishable activation kinetics, indicating that the gamma B catalytic region of C1s plays no role in the mechanism that triggers C1r activation in C1. The collagen-like fragments of C1q retained the ability to bind to C1s-C1r-C1r-C1s, but, in contrast with intact C1q, failed to induce C1r activation in the resulting complex at temperatures above 25 degrees C. On the basis of these observations it is proposed that activation of the serine-proteinase domain of C1r is controlled by a Ca(2+)-dependent intramolecular mechanism involving the Ca(2+)-binding alpha-region, and that this control is released in C1 by a signal originating in C1q and transmitted through the C1q/C1r interface.