We have characterized mutations induced in the SUP4-o gene of Saccharomyces cerevisiae by u.v. irradiation. Mutants were selected following treatment with 60 J/m2 u.v. light which reduced cell survival to 10% and increased the SUP4-o mutation frequency 100-fold above background. DNA sequence analysis of 120 mutants revealed that u.v. induced all types of base substitutions, although transitions, in particular G:C----A:T events predominated. In addition, a small number of single base pair deletions and double mutations, occurring in tandem or separated by a few base pairs, were recovered. The base pair substitutions were not distributed randomly in the SUP4-o gene and, with one exception, were all located at sites of adjacent pyrimidines, suggesting that they were targeted by u.v. photolesions. A substantial fraction of the mutations were detected at hotspots for u.v. mutagenesis. The majority of changes occurred at the 3' base of dipyrimidine sequences where both cyclobutane dimers and [6-4]-photoproducts could form. Approximately one-third of the induced base substitutions were found at potential pyrimidine dimer sites where [6-4]-photoproducts would be expected to occur rarely. The possible origins of the induced mutations and the role of cyclobutane dimers as premutational u.v. lesions in yeast are considered.