Normal rat kidney cells infected with the temperature-sensitive transformation mutant of Moloney murine sarcoma virus were used to study the biochemical and morphological changes that occur during transformation. The infected cells exhibited a normal morphology at the nonpermissive temperature (39 degrees C) and a transformed morphology at the permissive temperature (33 degrees C). A new viral protein was detected 2 hr after shift to the permissive temperature as a polyprotein with an estimated Mr of 85,000 (p85). Scanning electron microscopy of the cells within 5 hr after shifting them to the permissive temperature showed that they became smaller and rounded with numerous elongated microvilli. In an earlier study, changes in hexose uptake were found to occur 8-12 hr after the shift [Horn, J. P., Wood, T. G., Blair, D. G. & Arlinghaus, R. B. (1980) Virology 105, 516-525]. By 48 hr, the cells had the morphology of a fully transformed cell. Concomitant with the changes in the morphology were alterations in the cytoplasmic microtubule complex. At the nonpermissive temperature, the complex consisted of a lacy network of microtubules. Within 5 hr at the permissive temperature, the lacy network was still present but the microtubules were more diffusely stained and less discernible. By 48 hr, the microtubules were so diffuse that the lacy network could not be recognized. Alterations in the F-actin cables did not occur until 24 hr after shifting the cells to the permissive temperature. Enucleation of the cells at the nonpermissive temperature and shifting the cytoplasts to the permissive temperature did not result in the synthesis of detectable p85 or in any alteration of the cytoplast morphology or microtubule complex, suggesting that the temperature-sensitive lesion affects some event occurring in the nucleus.