The actin microfilament organization in rat embryo cells was examined by fluorescence microscopy with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and by electron microscopy, after mock infection or infection with adenovirus type 5 (Ad5). Infected cells showed severely reduced numbers of actin microfilaments and stress fibers, detectable early after infection. Mutants defective in Ad5 early genes were used to show that reduced microfilament organization was a function of the Ad5 transformation early gene 1a (E1a) and did not require expression of any other viral gene. The product of the E1a 13s mRNA was essential for the effect, although the 12s mRNA product appeared to contribute. Ad5 infection of the cells had no observable effect on total cell actin levels or on the ratio of monomeric to polymeric actin. E1a, therefore, affected only the higher-order organization of actin.