To study the relation between expression of the putative myofibroblast marker alpha-smooth muscle actin and the remodelling of extracellular matrix, immunocytochemical, gel electrophoresis, and collagen gel contraction studies were performed on two human fibroblast subtypes. Double immunolabelling for total actins and alpha-smooth muscle (sm) actin as well as affinity labelling of filamentous and monomeric actins in gingival fibroblasts demonstrated that alpha-sm was colocalized in stress fibres and in regions with high levels of monomeric actin throughout the cytoplasm. alpha-sm comprised up to 14% of total cellular actin as assessed by 2D gel electrophoresis. Thirteen different gingival and seven different periodontal ligament fibroblast lines constitutively expressed on alpha-sm actin. These cells exhibited up to 60% inter-line variations of fluorescence due to alpha-sm actin and up to 70% and 45% inter-line variation in the rate of collagen gel contraction. Quantitative, single cell fluorimetry of alpha-sm actin immunoreactivity demonstrated a linear relation between gel contraction and alpha-sm actin (correlation coefficients of 0.71 for gingival and 0.61 for periodontal ligament cells), but there was no detectable relationship between total actin content and gel contraction. In contrast, flow cytometry demonstrated that 99% of the total gated cells from cell lines exhibiting rapid gel contraction showed alpha-sm actin staining above background fluorescence as compared to only 35% of cells with slow rates of gel contraction. Contracting collagen gels stained with FITC-phalloidin showed cells with well-developed stress fibres that were progressively more compact and elongated during the time of maximal gel contraction. To examine the dependence of gel contraction on assembly of monomeric actin into actin filaments, cells were electroporated in the presence of phalloidin or cytochalasin D. Collagen gels exhibited up to 100% inhibition of gel contraction that was dose-dependent. Gel contraction was inhibited 93% by electroinjection of cells with alpha-sm actin antibody prior to incubation, but the antibody did not inhibit actin assembly after attachment and spreading on substrates. These data indicate that gel contraction is dependent on alpha-sm actin expression and that alpha-sm actin is a functional marker for a fibroblast subtype that rapidly remodels the extracellular matrix.