The specificity of expression of the isolated 5'-flanking sequences of the human beta- and epsilon-globin genes was examined in the K562 human erythroleukemia cell line, the murine erythroleukemia (MEL) cell, and in nonerythroid cell lines CV-1, HeLa-S3, and WI-38. Globin flanking sequences were active only in the erythroid K562 and MEL cell lines. Furthermore, the upstream sequence of the adult, beta-globin gene was active in MEL cells which express adult globin and not in K562 cells which express embryonic and fetal globins suggesting that tissue specificity resides in these upstream sequences. No expression was observed in K562 cells which have an embryonic-fetal hemoglobin phenotype.