Sixteen temperature-sensitive mutants of simian virus 40 were isolated after treatment of wild-type virus with UV light, nitrous acid, or nitrosoguanidine. All of the mutants were assigned to the B complementation group on the basis of quantitative complementation analyses. Characterization of several representative mutants revealed that under restrictive conditions such mutants produced immunologically identifiable tumor and viral capsid antigens, induced the synthesis of cellular DNA, and replicated infectious viral DNA. In addition, mutant particles produced at 33 degrees were more heat-labile than wild-type virus. This collection of mutants will be useful in an analysis of SV40 particle morphogenesis.