We have examined the expression of human alpha- and beta-like globin genes in transient heterokaryons formed by fusion of human nonerythroid cells with terminally differentiating mouse erythroleukemia (MEL) cells or with a MEL cell variant (GM979) in which the endogenous mouse embryonic beta-globin genes are activated. In both the parental MEL cells and the heterokaryons, the alpha-globin genes were activated at least 12 h earlier than the embryonic, fetal, and adult beta-globin genes. These results suggest that kinetic differences in the activation of alpha- and beta-like globin genes are not simply the result of different rates of accumulation of erythroid-specific regulatory factors but may reflect differences in the mechanisms governing the transcriptional activation of these genes during erythroid cell differentiation. In mouse GM979 x human nonerythroid heterokaryons, the human embryonic beta-globin gene was activated, consistent with our previous demonstration that erythroid cells contain stage-specific trans-acting regulators of globin gene expression. Moreover, a dramatic increase in the ratio of human fetal to adult beta-globin transcription was observed compared with that seen in MEL-human nonerythroid hybrids. This ratio change may reflect competition between the fetal and adult beta-globin genes for productive interactions with erythroid cell-specific regulatory elements. Finally, we demonstrate that the behavior of naturally occurring mutations that lead to aberrant hemoglobin switching in humans also leads to aberrant expression in transient heterokaryons. Therefore, erythroid cells must contain trans-acting factors that interact with mutated regulatory elements to induce high-level expression of the human fetal globin genes.