We have previously described stable mouse C127 cell lines in which a CaM mini-gene has been expressed in a bovine papilloma virus-based expression vector (Rasmussen and Means: EMBO J. 6:3961-3968, 1987). Elevation of CaM to levels five-fold higher than in control cells caused an acceleration in cell cycle progression by reducing the length of the G1 period. When these cell lines were originally isolated it was observed that cells in which CaM levels were increased had a flattened morphology. In this study we have examined the localization of actin, vimentin, and tubulin in these cells as compared to the BPV-transformed control cell line in order to determine if changes in shape were accompanied by differences in the cytoskeletal organization. Cell-cycle-dependent changes in the levels of mRNAs for histone H4, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, vimentin, and beta-tubulin have also been examined. Our results indicate that increased CaM causes differences in the organization of microfilaments, intermediate filaments, and microtubules and that these changes are accompanied by selective differences in the cell-cycle-dependent expression of some mRNAs. Elevated CaM was also correlated with a reduced stability of beta-tubulin mRNA. These studies indicate that CaM has pleiotropic effects on cell function and suggest that stable cell lines with altered CaM levels may provide a useful model system for understanding the molecular basis of CaM-dependent regulation of cellular processes.