Using a synthetic peptide mimicking the NH2-terminus of beta-actin we have raised a monoclonal antibody specific for this cytoplasmic actin isoform. Specificity of the antibody was demonstrated by its labelling of the actin polypeptide only in tissues containing the beta isoform, by its exclusive recognition of the synthetic beta-actin peptide amongst those mimicking all six vertebrate isoactins, and by its selective recognition of the beta-actin spot in two-dimensional electrophoresis gels of smooth muscle extracts. The antibody bound to actin filaments in both living and fixed fibroblasts where it labelled the stress fiber bundles and, more predominantly, the peripheral actin rich lamellipodia. The characteristics of the antibody indicate that it should serve as a useful tool for studying isoactin distribution and function.