Protein phosphorylation plays an important role in pheromone-induced differentiation processes of haploid yeast cells. Among the components necessary for signal transduction are the STE7 and STE11 kinases and either one of the redundant FUS3 and KSS1 kinases. FUS3 and presumably KSS1 are phosphorylated and activated during pheromone induction by a STE7-dependent mechanism. Pheromone also induces the accumulation of STE7 in a hyperphosphorylated form. This modification of STE7 requires the STE11 kinase, which is proposed to act before STE7 during signal transmission. Surprisingly, STE7 hyperphosphorylation also requires a functional FUS3 (or KSS1) kinase. Using in vitro assays for FUS3 phosphorylation, we show that pheromone activates STE7 even in the absence of FUS3 and KSS1. Therefore, STE7 activation must precede modification of FUS3 (and KSS1). These findings suggest that STE7 hyperphosphorylation is a consequence of its activation but not the determining event.