The major immediate-early (IE) genes 1 and 2 of human cytomegalovirus (HCMV) encode proteins that regulate the expression of HCMV genes as well as some other viral and cellular genes. In order to study the expression and function of these IE gene products, we established several HeLa cell lines that stably expressed the 68-kD IE1 protein, the 82-kD IE2 protein, or both proteins. The IE proteins expressed in these cell lines were biologically active, as shown by transient chloramphenicol acetyltransferase assays. Transcription from the major IE promoter was augmented in the IE1-expressing cells, while transcription from the HCMV early gene UL84 promoter was activated in the IE2-expressing cells. In addition, we found that the IE2-expressing cells established colonies in soft agarose more efficiently than the parental HeLa and the IE1-expressing cells. Furthermore, expression of both the IE1 and IE2 proteins was increased by treatment of these cell lines with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Thus, our cell lines provide a useful system to study the regulation of IE gene expression in human cells as well as to study transaction by HCMV IE proteins on various viral and cellular genes.