The cysteine-rich proteins (CRPs) are a family of highly conserved LIM (an acronym derived from the three gene products lin-11, isl-1 and mec-3) domain proteins that have been implicated in muscle differentiation. All CRP family members characterized so far have been shown to interact with the filamentous actin cross-linker α-actinin. The region of CRP required for this interaction has previously been broadly mapped to the molecule's N-terminal half. Here we report that the α-actinin-binding region of CRP, which we have mapped by using a combination of blot overlay and Western immunoblot techniques, is confined to an 18-residue sequence occurring within the protein's N-terminal glycine-rich repeat. A site-directed mutagenesis analysis of the binding region has revealed the critical importance of a single lysine residue (lysine 65 in human CRP1). Alterations at this site lead to a 10-fold decrease in α-actinin binding in comparison with wild-type CRP. The critical lysine residue localizes within a short α-helix, raising the possibility that mutagenesis-induced alterations in α-actinin-binding capacity might be attributed to the disruption of a key structural element.