AbstractM-CSF (or CSF-1) controls macrophage lineage development and function. A CSF-1-dependent culture system was established, which monitored the differentiation of CSF-1-responsive macrophage populations over time and upon adherence. Wiskott-Aldrich syndrome protein verprolin homologous (WAVE) proteins are involved in actin reorganization, a process critical to many cell functions. WAVE2 but not WAVE1 has been considered significant for macrophage function. Using the CSF-1-dependent differentiation system, we were able to demonstrate the contrasting regulation of the expression of WAVE1 and WAVE2; the levels of the latter rose over time and as the macrophage population became adherent, although those of the former increased over time but were down-regulated upon adherence. Evidence was obtained that WAVE1 was also cleaved to a novel, 60-kDa fragment by macrophage adherence and by another pathway involving calpain-mediated proteolysis. Mutagenesis studies indicated that cleavage of WAVE1 by calpain results in the removal of the verprolin-homology, cofilin-like, and acidic domain and thus, the loss of WAVE1 activity. We suggest that WAVE1 is also important for macrophage biology and that it could have separate functions to those of WAVE2.