Somatic complementation by fusion of two mutant cells and mixing of their cytoplasms occurs when the genetic defect of one fusion partner is cured by the functional gene product provided by the other. We have found that complementation of mutational defects in the network mediating stimulus‐induced commitment and sporulation of Physarum polycephalum may reflect time‐dependent changes in the signaling state of its molecular building blocks. Network perturbation by fusion of mutant plasmodial cells in different states of activation, and the time‐resolved (...) analysis of somatic complementation effects can be used to systematically probe network structure and dynamics. Time‐resolved somatic complementation quantitatively detects regulatory interactions between the functional modules of a network, independent of their biochemical composition or subcellular localization, and without being limited to direct physical interactions. BioEssays 25:950–960, 2003. © 2003 Wiley Periodicals, Inc. (shrink)