Actins comprise six isoforms of which the nonmuscle isoforms -/-actins are expressed by all eukaryotic cells. The expression pattern of one of the muscle actin isoforms, -sm actin, previously believed to be restricted to smooth muscle, has been broadened to encompass activated fibroblasts (myofibroblasts) as well. The significance of this molecular conversion has remained largely unknown. We have recently shown that a reduction in filamentous -sm actin by electroinjected specific antibodies or antisense oligodeoxynucleotides leads to increased motility in breast myofibroblasts (Rønnov-Jessen L., Petersen, O. W. J. Cell Biol. 1996, 134, 67-80). In the present study we have expanded on the functional significance of actin isotypes in fibroblasts from the opposite point of view, namely filamentous nonmuscle actin. Nonmuscle actins in fibroblasts and myofibroblasts were ADP-ribosylated by Clostridium botulinum C2 toxin. The substrate for C2 toxin is globular actin, which upon ribosylation cannot incorporate into microfilaments. The pattern of actin ADP-ribosylation in (myo)fibroblasts in the presence of [32P]NAD was analyzed by isoelectric focusing, fluorography and immunoblotting. The influence of C2 toxin on microfilaments in intact cells was further assessed by immunofluorescence, and motility was measured in a mass migration assay and by computerized video time-lapse microscopy. We show here that C2 toxin specifically ribosylates - and -actin in both fibroblasts and myofibroblasts. Whereas fibroblasts rapidly round up and stop migrating when filamentous -/-actin is reduced by short-term ADP-ribosylation, myofibroblasts maintain their flattened morphology and a basic low motility.