Invasion in gut epithelial cells, mediated by genes of the Salmonella pathogenicity island I, is a crucial step in the pathogenesis of Salmonella enterica serovar Enteritidis infections. The most important regulator of the invasive process is the hilA gene. In this study, a transposon bank approach was used to identify DNA sequences affecting expression of hilA. Mutants with decreased hilA expression carried mutations in known virulence gene regulators (fliZ, hilD, sirA), genes encoding ion transport proteins (feoA, feoB, pstB, pstC), genes involved in transcription/translation machinery (nusA, selA) and the hypothetical inner membrane protein STM2303. Mutants yielding increased hilA expression carried a transposon insertion in the known virulence regulator hha, the transcriptional regulator and oxygen sensor fnr and the virulence gene virK. Mutants having decreased and increased hilA expression were more and less invasive in the human colon carcinoma cell line T84 compared to wild type strain bacteria, respectively.