The expression of MMP-9 in rat embryo fibroblasts (REF) transformed with Ha-ras or with Ha-ras and v-myc is associated with metastatic behavior. In contrast, REF transformed with Ha-ras and the adenovirus EIA genes (EIA) are tumorigenic, do not release MMP-9 and are rarely metastatic. In this report, we establish that EIA expression results in decreased levels of MMP-9 mRNA in an Ha-ras and v-myc transformed cell line and examine which of the functional domains of EIA participate in the inhibition of MMP-9 expression and which contribute to the suppression of metastasis. The metastatic 2.10 REF line, derived by co-transfection with v-myc and Ha-ras, constitutively expresses high levels of MMP-9 (92 kDa gelatinase). Transfection of E1A wild-type plasmids into this cell line eliminates detectable MMP-9 mRNA expression and greatly reduces MMP-9 activity. Transfection of 2.10 with E1A plasmids encoding mutations in conserved region 2 (CR2) retained inhibition of MMP-9 similar to the inhibition seen with wild-type E1A. Transfection with E1A containing mutations in CRI or the amino terminal region diminished, but did not fully inhibit, MMP-9 expression. In contrast, inhibition of MMP-9 was lost in with E1A mutations in CR3. Cells transfected with E1A mutants in CRI, the amino terminal region or CR3 retained metastatic behavior. Our Findings delineate the regions of E1A responsible for MMP-9 inhibition and further define the domains of E1A responsible for inhibition of metastasis.