We have constructed yeast vectors in which derivatives of the adenovirus E1A gene are expressed from the GAL1 promoter. Cells expressing E1A289 grow poorly and accumulate cells with a 1C DNA content. Using a series of E1A deletion mutants, we have identified three regions within the E1A protein that are necessary for the G1 growth phenotype; each deletion partially relieves the growth defect. These deletions span residues 4-25, 38-60 and 140-186, which fall within the N-terminal, CR1 and CR3 domains of E1A respectively. Expression of the first 82 residues of E1A, spanning just the N-terminal and CR1 domains, strongly inhibits yeast cell growth in G1 showing that these domains can function independently of other domains of E1A. Using this strong growth inhibition, we isolated a yeast mutant in the net1 gene that conferred resistance to the expression of E1A1-82. The mutant was insensitive to expression of both E1A1-82 and full length E1A, but remained sensitive to the toxicity caused by over-expression of a Gal4p-VP16 fusion. Finally, we found that the function of E1A in yeast depends on the cyclic AMP signaling pathway, providing a striking parallel with the action of E1A at the c-fos promoter in mammalian cells. These results suggest that a genetic analysis of the yeast model system will provide relevant new insights into mechanisms of gene regulation by E1A proteins.