The structural gene encoding human delta-aminolevulinate dehydratase has been assigned to the long arm of chromosome 9 by somatic cell hybridization techniques using murine erythroleukemia-human fibroblast somatic cell hybrids. Dimethyl sulfoxide induction of erythroid differentiation in these hybrid cells resulted in a 3 to 12-fold increase in the levels of total delta-aminolevulinate dehydratase. Human delta-aminolevulinate dehydratase was detected by an immunodiscrimination assay using polyclonal mouse anti-human delta-aminolevulinate dehydratase antibodies. Of four primary hybrid clones, each from an independent fusion, one hybrid line, XX-8, was positive for human delta-aminolevulinate dehydratase. Examination of 23 secondary, tertiary, and quaternary XX-8 subclones revealed that the expression of the human isozyme segregated with human chromosome 9q, confirming the provisional regional assignment made by classical linkage studies. One positive quaternary clone, XX-8-H21-H7-2, expressed human delta-aminolevulinate dehydratase activity and contained only human 9q13----qter. In addition, studies of tertiary and quaternary subclones from two series, XX-8-A31 and XX-8-H21-H7, indicated that murine regulatory factors increased the human as well as the murine enzymatic activity following induction of erythroid differentiation.