In vivo, alpha-smooth muscle actin (SMA) is expressed de novo and temporarily by fibroblastic cells during wound healing and correlates particularly with wound contraction. In culture, the presence of varying proportions of cells expressing and not expressing this actin isoform (alpha-SMA-positive and alpha-SMA-negative cells) is characteristic of fibroblastic populations from different tissues. It is possible that mechanisms controlling the expression of actin isoforms, and thus modulating cytoskeleton-related functions, play a major role in the organization of cell shape and motility. We have compared the cell shape as well as the cytoskeleton and focal contact organization in alpha-SMA-positive and alpha-SMA-negative rat fibroblasts from various organs (i.e., skeletal muscle, dermis, subcutaneous tissue, and lung). Within each category, i.e., alpha-SMA-positive or alpha-SMA-negative fibroblasts, no significant morphological differences were seen among populations derived from different tissues. In contrast, alpha-SMA-positive and alpha-SMA-negative fibroblasts were significantly different, independently of their origin: alpha-SMA-positive cells had larger average areas, higher numbers of narrow extensions at the edges, larger focal adhesions with the substratum, and a more important network of cellular fibronectin than alpha-SMA-negative cells. Thus, alpha-SMA-positive and alpha-SMA-negative variants naturally present in fibroblastic populations exhibit important phenotypic differences probably associated with distinct functional activities.