K562 human erythroleukemia cells can be induced by hemin and other stimuli to produce hemoglobin and actively express epsilon- and gamma-globin genes but not the adult-like beta-globin gene. The control of globin gene expression is regulated by both cis-acting DNA sequences, such as the 5'-promoter region and 3'- and 5'-enhancers, and trans-acting factors. The activation of 5'-promoter DNA sequences mediated by trans-acting factors can be examined in a cell-free system by in vitro transcription. We report here the differential transcriptional activation of globin genes by nuclear extracts from K562 cells without the need of supplemental HeLa whole cell extracts. Transcription of the epsilon-globin gene which is more active in K562 cells than the gamma-globin gene was greater at low nuclear extract concentrations (protein concentration less than 1.5 mg/ml) than the gamma-globin gene. However, neither beta-globin nor insulin promoters (which are not active in intact K562 cells) were active in the nuclear extract in vitro transcription system. The accurate initiation and specific transcription of globin genes by K562 nuclear extracts suggest that trans-acting factors interacting with the 5'-promoter gene sequence regulate in part the extent and specificity of gene expression and that the activity of these factors can be preserved during their isolation.