Thymidine kinase (TK) was isolated from four different cell lines (TK +/+ P4, TK +/- P4.3.4, TK +/- 3.7.2C, and TK -/- P4.3) that represent the genotypes of L5178Y mouse lymphoma cells. TK isolated from each of the different cell lines was characterized with respect to the estimated isoelectric point (pI), temperature sensitivity, estimated substrate dissociation constants (Km), estimated inhibitor constant (Ki), and response to the activator deoxycytidine diphosphate. The characteristics of TK from the different cell lines were compared to determine whether the product of the TK gene was changed by the mutation that produced the TK -/- genotype and reverse mutation to the TK +/- genotype. The results indicate that the TK enzymes isolated from the TK +/+ P4, TK +/- P4.3.4, and TK +/- 3.7.2C cells have similar, if not the same, characteristics. The small amounts of TK associated with TK -/- P4.3 and the mitochondria from TK +/+ P4 had similar characteristics, and both were different in many respects from the TK associated with the TK +/+ P4, TK +/- P4.3.4, and TK +/- 3.7.2C. These results raise a question about whether a structural gene is the target for chemical mutagens in the L5178Y TK +/- assay; however, this can only be answered by the isolation of the TK gene from each of the L5178Y genotypes and determination of the nucleotide sequence.