Randomly growing Chinese hamster lung cells were pulse-labeled with 3H-thymidine, and the replicating forks of individual DNA fibers were visualized by autoradiography. When grown in complete medium, wild-type SV40-transformed cells had more forks per unit length of DNA than nontransformed cells. In isoleucine-depleted medium, wild-type SV40-transformed cells had fewer forks per unit length than those few nontransformed cells (1-3% of the population) which continued DNA replication. Cells transformed by a tsA mutant of SV40 when grown at the permissive temperature had more forks per unit length in complete medium and fewer forks per unit length in depleted medium than nontransformed cells, but when grown at the restrictive temperature, the tsA-transformed cells behaved like nontransformed cells.