1. Biochemical and quantitative image analysis methods were used to investigate the anatomical basis for the previously described agonist-induced redistribution of calponin. 2. At 140 nm resolution, the quantitative distribution of calponin in resting cells was statistically indistinguishable from that of filament bundles containing alpha-smooth muscle actin and myosin, but was significantly different from that of filaments containing beta-non-muscle actin. Conversely, in stimulated cells, the distribution of calponin was not significantly different from that of beta-actin filaments in the subplasmalemmal cell cortex but was significantly different from the distribution of alpha-actin- and myosin-containing filamentous bundles. 3. The distribution of calponin significantly differed from that of the intermediate filament proteins vimentin and desmin as well as that of the dense body protein alpha-actinin either by ratio analysis of the subcellular distribution or by colocalization analysis. 4. The imaging results, although limited to 140 nm spatial resolution, suggested the hypothesis that the agonist-induced redistribution involves the binding of calponin to isoform-specific actin filaments. This hypothesis was tested by quantifying the relative affinity of calponin for purified alpha- and beta-actin. Light scattering measurements showed that calponin induces bundle formation with beta-actin more readily than alpha-actin, indicating that calponin may be preferentially sequestered by beta-actin under appropriate conditions. 5. These results are consistent with a model whereby agonist activation decreases calponin's binding to filaments, but the tighter binding to beta-actin filaments results in a spatial redistribution of calponin to the submembranous cortex.