High mobility group (HMG) 14 is a ubiquitous chromosomal protein that binds specifically to nucleosomal DNA and may be involved in a process that confers distinct properties to the chromatin structure of transcriptionally active genes. To explore the involvement of this protein in regulation of gene expression, we studied the effect of aberrant expression of HMG-14 protein on cellular differentiation. We produced stably transfected C2C12 mouse myoblasts expressing the human HMG-14 protein under the control of the mouse mammary tumor virus promoter. Transformed colonies retained their potential do differentiate into myotubes. Induction of human HMG-14 expression by dexamethasone inhibited the myogenic process. Revertant colonies, which lost the ability to express human HMG-14, regained the ability to differentiate into myotubes. Inhibition of myoblast differentiation by aberrantly expressed HMG-14 correlated with down-regulation of myogenic determination factors. The results suggest that proper cellular differentiation requires regulated expression of HMG-14 protein and are consistent with the possibility that this protein may be involved in gene regulation.