Actin-fragmin is a heterodimeric protein complex from Physarum polycephalum microplasmodia that is phosphorylated in vitro at residues Thr203 and Thr202 of the actin subunit by the endogenous actin-fragmin kinase. Following phosphorylation, the F-actin capping activity of the complex becomes Ca(2+)-dependent, suggesting a fundamental regulatory role in controlling F-actin growth [Gettemans, J., De Ville, Y., Waelkens E. and Vandekerckhove, J. (1995) J. Biol. Chem. 270, 2644-2651]. In this study we analysed actin phosphorylation in vivo. We demonstrate that the actin-fragmin complex constitutes the only substrate of the actin-fragmin kinase in plasmodia. Monomeric actin is not phosphorylated. Immunoprecipitation of actin-fragmin reveals that approximately 40% of the actin subunit of the complex is phosphorylated in vivo. However, using purified substrate and kinase, the complex can be quantitatively phosphorylated as judged by two-dimensional gel electrophoresis. Through comparative phosphopeptide fingerprinting, we show that the phosphorylation sites in vivo are identical to those identified in vitro. We additionally characterized a complex of actin and the NH2-terminal half of fragmin (residues 1-168) that is also phosphorylated by the same kinase. In contrast to actin-fragmin, phosphorylation of the complex between actin and residues 1-168 of fragmin is independent of Ca2+ because the second Ca(2+)-dependent regulatory actin-binding domain is missing. By artificially varying the actin-fragmin concentration or the actin-fragmin kinase activity present in microplasmodia cytosolic extracts, we attempted to detect alternative protein substrates for the actin-fragmin kinase. The fact that none could be identified suggests that the control and properties of actin-fragmin phosphorylation observed in vitro may stand as a model for F-actin growth control in Physarum cells.