Salmonella typhi, the causative agent of human typhoid fever, has been shown to trigger a (cytochalas-ininhibited) microfilament-dependent mechanism of entry into human epithelial cell lines. Using an in vitro internalization assay in which bacteria are incubated with a young, confluent INT407 monolayer for 1 hr followed by 1 hr in the presence of growth media containing gentamicin, we have analyzed bacterial uptake efficiency over a wide range of MOI's (i.e. from 0.4 to 4000) and studied the response to a variety of biochemical inhibitors and antibodies. S. typhi invasion efficiency {(# of bacteria internalized at end of assay/starting inoculum#)x 100 = % invasion} increased steadily to a maximum at an MOI of ~40 and decreased sharply thereafter, suggesting that invasion entry sites are saturated at these higher MOI's. Acridine orange staining of infected cells over time revealed that there are approximately 34 physical entry sites per INT407 apical cell surface. Further studies have shown that these entry sites are exhausted at higher MOI's after 30-45 minutes and only minimal S. typhi invasion can occur.