The major transforming activity of polyomavirus, middle T antigen, targets several cellular regulatory effectors including protein phosphatase 2A and src tyrosine kinases. Although transformed cells exhibit profound morphological changes, little is known about how middle T antigen‐induced changes in the cellular regulatory environment specifically affect the cytoskeleton. We have investigated these changes in 10T½ mouse fibroblasts transformed with polyoma middle T antigen. Immunofluorescence microscopy revealed that expression of middle T antigen (Pym T cells) depleted the stable (acetylated) microtubule array and increased the sensitivity of dynamic (tyrosinated) microtubules to nocodazole‐induced disassembly. These effects were associated with a modest but statistically significant (P≤0.05) increase in recovery of protein phosphatase 2A activity with microtubules. Middle T antigen expression also depleted the normal cellular complement of actin stress fibers and focal adhesions, in parallel with changes in the distribution of src tyrosine kinases. Herbimycin A promoted recovery of paxillin and phosphotyrosine into nascent focal adhesion sites, in addition to restoring normal src tyrosine kinase distribution. However, herbimycin A did not restore actin stress fibers or parental‐type microtubules to Pym T cells. We suggest that regulation of the microtubule array by middle T antigen may occur through direct effects including redistribution of protein phosphatase 2A as well as indirect effects such as altered interactions with actin‐based stress fibers. Cell Motil. Cytoskeleton 47:253–268, 2000. © 2000 Wiley‐Liss, Inc.