During primary culture, arterial smooth-muscle cells (SMCs) undergo transition from a contractile to a synthetic phenotype. As a consequence, they lose the ability to contract and, instead, acquire the ability to synthesize DNA, divide and produce extracellular-matrix components. In the present study, we used cytochemical and electron-microscopic methods to study the organization of the cytoskeleton in primary cultures of adult rat and human arterial SMCs. Freshly isolated cells were all in contractile phenotype and stained intensely with NBD-phallacidin, a fluorescent marker for F-actin. Diffuse, positive staining was also obtained using indirect-immunofluorescence microscopy with antibodies against tubulin and vimentin, which are subunit proteins of microtubules and intermediate filaments, respectively. Fine structurally, the cytoplasm of these cells was mainly filled with microfilament bundles coalescing in dense bodies. After a few hours in culture, the SMCs attached to the substrate and started to extend processes in various directions. These stained with antibodies to tubulin and vimentin, but not with NBD-phallacidin. Within 1-3 days of culture, the cells spread out on the substrate and developed a system of actin-containing stress fibre bundles spanning their entire length, as well as a radiating system of microtubules and vimentin filaments, originating in the juxtanuclear region. Fine structurally, these changes corresponded to a marked decrease in the number of microfilaments, an increase in the number of microtubules and intermediate filaments, and the formation of an extensive rough endoplasmic reticulum and a large Golgi complex. The morphological transformation of the cells was accompanied by the coordinated activation of DNA, RNA and protein synthesis.