Smooth muscle cell calponin (h1 or basic isoform) is an actin-binding protein that inhibits actomyosin MgATPase activity and is abundantly expressed in differentiated smooth muscle. Western blots showed bovine tracheal (BT) smooth muscle cells in culture expressed only 2 +/- 1% (n = 8) of the amount of calponin in tissues, while NIH-3T3 fibroblasts expressed none. We tested the hypothesis that introduction of calponin to cultured BT and 3T3 cells would inhibit cytoskeletal activities associated with cell proliferation. To achieve high-efficiency expression, an adenovirus encoding the CMV-calponin construct (Adv-CaP) was generated by homologous recombination in 293 cells. With greater than 90% of BT and 3T3 cells infected with Adv-CaP, calponin expression (32 and 11 microg/mg total protein, respectively) was similar to that in smooth muscle tissues (51 microg/mg). Cells were infected with Adv-CaP for 48 h, replated at low density and proliferation rates were assessed by cell density and [3H]thymidine incorporation. Cell growth and DNA synthesis by Adv-CaP-infected cells were inhibited to one-third control values for both BT and 3T3 cells. Expressed calponin was localized primarily on stress fibers in both cell types. Calponin may act at the cytoskeletal level to retard signaling pathways that normally lead to tight coupling between cell shape and DNA synthesis.