The tyrosine kinase ZAP-70 plays a critical role in signal transduction in T cells and NK cells but has limited expression in primary human B cells. ZAP-70 is, however, expressed in adult B cell chronic lymphocytic leukemia where it correlates with a poor prognosis. We wished to determine if ZAP-70 is also expressed in pediatric B cell malignancy. A quantitative PCR assay for ZAP-70 expression was established and ZAP-70 expression in a range of human B cell lines was compared with expression in the Jurkat T cell line. ZAP-70 expression was then determined in bone marrow lymphoblasts obtained from 12 patients with pre-B cell acute lymphoblastic leukemia (ALL). ZAP-70 expression was not detected in mature B cell lines but was detected in pre-B cell lines at a level comparable to that seen in T cells. ZAP-70 expression was strongly expressed in nine of the 12 cases of primary pre-B cell lymphoblastic leukemia. The T cell-associated protein kinase ZAP-70 is highly expressed in pre-B lineage cells and most cases of pre-B acute lymphoblastic leukemia. ZAP-70 expression may hold prognostic value for pre-B ALL and raises the prospect of a novel therapeutic target.