Frozen or paraffin-embedded human and rat lung specimens were stained with antibodies against total actin, alpha-smooth muscle (SM) actin, vimentin, desmin, or gelsolin. Alveolar interstitial myofibroblasts [i.e., contractile interstitial cells (CIC)] were labeled by total actin antibody but not by alpha-SM actin antibody. They stained for vimentin and gelsolin and, in rat lungs, most of them for desmin. Pericytes located around venules at the junction of three alveolar septa were always positive for alpha-SM actin and never for desmin. Tissue samples were also immunostained by an alpha-SM actin antibody and studied by electron microscopy. With this technique we confirmed that cells, identified as pericytes on the basis of their location, were intensely labeled by alpha-SM actin antibodies, whereas alveolar myofibroblasts were not. We conclude that in the lung interstitium pericytes and alveolar myofibroblasts have distinct cytoskeletal features, alpha-SM actin antibody staining being a simple method to distinguish between them. Furthermore, it appears that alveolar myofibroblasts have a peculiar pattern of cytoskeletal protein composition which, in the rat, is similar to that previously described for stromal cells in uterine submucosa, liver sinusoids (Ito cells), or the core of intestinal villi.