The cloaca acts as a common chamber into which gastrointestinal and urogenital tracts converge in lower vertebrates. The distal end of the cloaca is guarded by a ring of cloacal muscles or sphincters, the equivalent of perineal muscles in mammals. It has recently been shown that the development of the cloacal musculature depends on hindlimb muscle formation. The signaling molecules responsible for the outward migration of hindlimb myogenic precursors are not known. Based on the expression studies for CXCR4 and SDF-1, we hypothesized a role of this signaling pair during cloacal muscle precursor migration. The aim of our study was to investigate the role of SDF-1/CXCR4 during cloacal muscle precursor migration in the chicken embryos. We show that SDF-1 is expressed in the cloacal region, and by experimentally manipulating the SDF-1/CXCR4 signaling, we can show that SDF-1 guides the migration of CXCR4-expressing cloacal muscle precursors. Developmental Dynamics 239:1622–1631, 2010. © 2010 Wiley-Liss, Inc.