Three new kinds of recombinant DNA constructs were used to transfer cloned human class I HLA genes (A2 and B8) into unique HLA mutant lymphoblastoid cells: (i) pHeBo(x): a class I gene, 'x,' in plasmid ventor pHeBo, which contains a hygromycin resistance gene and Epstein-Barr virus oriP element that sustains extrachromosomal replication; (ii) pHPT(x): gene x in a vector with a hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene; (iii) pHPTe(x): gene x in a vector with the HPRT gene and oriP element. Cell surface class I antigen expression was strong in transferents made with class I-deficient lymphoblastoid cell line mutants .144 (A-null), .53 (B-null), and .184 (A-null, B-null). Transferents expressing HLA-A2 were recognized specifically by HLA-A2-specific cytotoxic T lymphocytes. When introduced on either of the vectors with the Epstein-Barr virus oriP element, the class I gene replicated extrachromosomally and was lost at rates of 0.2 to 0.3 per cell division. When introduced with vector pHPT (lacking Epstein-Barr virus oriP), the B8 gene was inserted at different chromosomal locations. Introduction of the HLA-B8 gene failed to restore antigen expression by HLA-B-null mutant .174, providing evidence that, unlike mutants exemplified by .53, .144, and .184, some HLA antigen loss mutants are deficient in a trans-acting fuction needed for class I antigen expression. Of more general interest, the results obtained with HLA class I genes in vectors that replicate extrachromosomally suggest ways of relating genic expression to chromatin structure and function and of attempting to clone functional human centromeres.