ABSTRACT A medium containing chlorolactate has been devised to enrich for mutants that are unable to utilize lactate for growth, and therefore that may be defective in cytochrome c. Complementation tests of 6,520 chlorolactate-resistant mutants that were obtained spontaneously or induced with UV, ICR-170, or nitrosoimidazolidone resulted in the identification of 195 mutations at the cycl locus, which controls the primary structure of iso-1-cytochrome c. These 195 mutants, with 16 cycl mutants previously isolated, were examined for total cytochrome c by spectroscopic methods, growth on lactate medium, suppressibility by defined nonsense suppressor, mutational sites by x-ray-induced recombination, ability to revert, and in 86 cases, whether intragenic revertants contain altered iso-1-cytochrome c. Except for the deletion mutant cycl-1, all of thb mutants appeared to contain single-site mutdons that could be assigned to at least 35 different sites within the gene. The cycl mutants either completely lacked iso-1-cytochrome c or contained iso-1-cytochromes c that were completely or partially nonfunctional. In spite of the fact that the cyd mutants obtained by the chlorolactate procedure were selected on the basis of defective function, 68% appeared to completely lack iso-1-cytochrome c. The remaining cycl mutants contained below normal amounts of iso-1-cytochromes c. Studies at several incubation temperatures indicated that these nonfunctional iso-1-cytochromes c were thermolabile. It is suggested that the predominant means for abolishing iso-1-cytochrome c by mutations are either through a complete loss, such as produced by chain terminating codons, or impairments through drastic changes of tertiary structure which lead to instability and thermolability.