Plasticity of smooth muscle alpha-actin gene expression in fibroblasts and vascular smooth muscle cells is mediated by opposing effects of transcriptional activators and repressors. Among these factors, three single-stranded DNA-binding proteins, Puralpha, Purbeta, and MSY1, have been implicated as coregulators of a cryptic 5'-enhancer module. In this study, a molecular analysis of Purbeta, the least well characterized member of this group, was conducted. Southwestern and Northwestern blotting of purified Purbeta deletion mutants using smooth muscle alpha-actin-derived probes mapped the minimal single-stranded DNA/RNA-binding domain to a conserved region spanning amino acids 37-263. Quantitative binding assays indicated that the relative affinity and specificity of Purbeta for single-stranded DNA were influenced by purine/pyrimidine content; by non-conserved regions outside amino acids 37-263; and by cell-derived proteins, specifically MSY1. When overexpressed in A7r5 vascular smooth muscle cells, Purbeta (but not Puralpha) inhibited transcription of a smooth muscle-specific mouse alpha-actin promoter transgene. Structural domains required for Purbeta repressor activity included the minimal DNA-binding region and a C-terminal domain required for stabilizing high affinity protein and nucleic acid interactions. Purbeta inhibitory activity in transfected A7r5 cells was potentiated by MSY1, but antagonized by serum response factor, reinforcing the idea that interplay among activators and repressors may account for phenotypic changes in smooth muscle alpha-actin-expressing cell types.