Gene targeting provides a powerful means for analyzing gene function, as exemplified by knockout mouse studies and recent work with the highly recombinogenic chicken DT40 B-lymphocyte line. In human cultured cells, however, the low frequency of gene targeting is a serious barrier to efficiently generate knockout clones. Moreover, commonly used human cell lines are karyotypically abnormal or unstable. Here, we show using promoterless targeting constructs that Nalm-6, a human pre-B ALL cell line, is highly proficient for gene targeting by homologous recombination. Indeed, the efficiency of TP53 gene targeting in Nalm-6 appears nearly two orders of magnitude higher than that in HCT116, a colon cancer cell line popularly used for gene targeting. Expression analysis revealed a lack of MSH2 expression in this cell line. As Nalm-6 has a stable neardiploid karyotype with normal p53 status, our results underscore the usefulness of Nalm-6 for gene knockout studies in humans.