A 980-bp subfragment of human cytomegalovirus (HCMV) strain Towne has been previously identified as morphologic transforming region II (mtrll) because of its ability to induce focal transformation of NIH 3T3 cells. Transcripts from this region, which could encode the three open reading frames (ORFs), 79, 83, and 34 amino acids (aa), detected by DNA sequence analysis, are expressed early during HCMV infection. In this report, the mRNA start sites for promoters (PI and P2) were mapped within Towne mtrll by primer extension using RNAs isolated from transformed NIH 3T3 cells. The Towne mtrll promoters exhibited similar activities to the SV40 enhancerless early promoter. Equivalent promoter activities were detected within the mtrll colinear nontransforming region from HCMV strain Tanaka. Two subclones of Towne mtrll (5’ 440-bp and 3’ 540-bp), each containing one promoter, were generated utilizing a unique BgII site which also interrupted the 79-aa ORF. In transfection assays, neither the 5’ 440-bp promoter subclone containing a truncated 79-aa ORF nor the 3’ 540-bp subclone containing intact 83- and 34-aa ORFs exhibited transforming activity. These data indicated that transformation by HCMV mtrll did not occur by promoter insertion. The identification of these early promoters will allow further studies on the regulation of important HCMV early genes known to be involved in viral/host inter