Members of the cysteine-rich protein (CRP) family are evolutionarily conserved proteins that have been implicated in the processes of cell proliferation and differentiation. In particular, one CRP family member has been shown to be an essential regulator of cardiac and skeletal muscle development. Each of the three vertebrate CRP isoforms characterized to date is composed of two copies of the zinc-binding LIM domain with associated glycine-rich repeats. In this study, we have addressed the biological significance of the CRP multigene family by comparing the subcellular distributions, biochemical properties, and expression patterns of CRP1, CRP2, and CRP3/MLP. Our data reveal that all three CRP family members, when expressed in adherent fibroblasts, associate specifically with the actin cytoskeleton. Moreover, all three CRP isoforms are capable of interacting with the cytoskeletal proteins alpha-actinin and zyxin. Together, these observations suggest that CRP family members may exhibit overlapping cellular functions. Differences between the three CRPs are evident in their protein expression patterns in chick embryos. CRP1 expression is detected in a variety of organs enriched in smooth muscle. CRP2 is restricted to arteries and fibroblasts. CRP3/MLP is dominant in organs enriched in striated muscle. CRP isoform expression is also developmentally regulated in the chick. Our findings suggest that the three CRP family members perform similar functions in different muscle derivatives. The demonstration that all members of the CRP family are associated with cytoskeletal components that have been implicated in the assembly and organization of filamentous actin suggests that CRPs contribute to muscle cell differentiation via effects on cytoarchitecture.