Multistate outbreaks of Escherichia coli O157:H7 infection in 2005 and 2006 associated with fresh and especially minimally processed produce greatly escalated the application of rapid pathogen detection systems to safety management in this food category. Pathogen testing was rapidly integrated into preharvest qualification for field lots, incoming raw produce, or final product. The raw produce and final product were incorporated into test-and-hold programs, typically within a 10-h time frame. To enhance consumer safety and provide guidance for the industry, an assessment of selected kits in comparison to a culture-based method was undertaken. Four primary kits were compared: the Neogen Reveal, SDI RapidChek, BioControl GDS O157, and Qualicon BAX O157 MP. Nine different leafy greens were freshly harvested and inoculated with a five-isolate mixture of E. coli O157:H7 at 10 CFU/25 g of sample, and cultures were enriched following the specified protocol. The PCR method was most consistent for identifying the presence of the inoculated pathogen in the shortest period of time. For the red-pigmented leafy vegetables red butter lettuce, curly endive, red lettuce, and lollo rosa, 13, 38, 88, and 100% false-negative results, respectively, were obtained with the immunoassays, but PCR detection was minimally affected. Immunoassays were negatively affected by delays in achieving critical threshold populations during the allowed enrichment period. Leafy green type, temperature abuse, and preharvest environment were unlikely to affect the results of PCR-based kits. Findings strongly suggest that product testing systems using 8-h detection cutoffs may give false-negative results. These issues become very important in high-throughput testing and retest protocols for presumptive pathogen-positive lots of produce.