Egr-1 is an early growth response gene that encodes a protein with three zinc fingers and is involved in transcriptional regulation. In adult heart myocytes, in contrast to c-fos and c-myc, high levels of Egr-1 mRNA expression have been shown. Here we report that Egr-1 transactivates rat cardiac alpha-MHC gene expression. In serum-starved primary cultures of 18-day-old fetal rat heart myocytes, addition of serum evoked expression of both Egr-1 and alpha-MHC gene transcripts. Inclusion of 10 microM cycloheximide in these cultures for 48 h caused a greater increase in Egr-1 mRNA, whereas the expression of alpha-MHC transcripts was ablated. To examine the involvement of Egr-1 in alpha-MHC induction, we transfected primary cultures of cardiac myocytes with plasmids pCMVEgr-1 (Egr-1 expression vector) and pMP3.3CAT containing -2.9- to +0.42-kilobase sequences of the alpha-MHC gene fused to the coding region of the chloramphenicol acetyltransferase (CAT) gene. Cotransfection of pCMVEgr-1 stimulated expression of pMP3.3CAT 10-15-fold. Furthermore, pCMVEgr-1 also stimulated expression of the endogenous alpha-MHC gene in primary cultures of cardiac myocytes. Transactivation of pMP3.3CAT expression by pCMVEgr-1 was also observed by transfecting the myogenic cell line Sol 8, but not in L6E9 cells or in NIH3T3 fibroblasts. By creating progressive 5' deletions of the alpha-MHC gene, we found that the region extending between -1698 and -1283 base pairs is necessary for Egr-1-induced expression of the alpha-MHC/CAT construct. These results define a physiological target for the Egr-1 transcription factor and delineate a novel mechanism for regulation of the alpha-MHC gene.