In this report, we describe the cloning of the human cytoplasmic thymidine kinase (tk-C; EC 2.7.1.21) gene and its preliminary characterization. The tk-C sequences were isolated from a phage genomic library made from DNA of a transfected mouse cell carrying the human tk-C gene. The human transforming sequences were identified by homology with human Alu sequences. Six recombinant phages were isolated and five were competent to transfer human TK-C activity to TK-deficient mouse cells when transferred in pairs. Conclusively, sequences homologous to these clones are present in all human TK+ transformants examined. We estimate the maximal size of the tk-C gene to be 14 kilobase pairs and its minimal size to be between 4 and 5 kilobase pairs. The gene contains many noncoding inserts and numerous Alu sequences.