Aims: To determine survival and colonization of Escherichia coli O157:H7 on spinach leaves as affected by inoculum level and carrier, temperature and relative humidity (r.h.). Methods and Results: Spinach leaves were inoculated with suspensions of E. coli O157:H7 in distilled water (DW) and 0.1% peptone water (PW) and incubated at 4, 12 and 25 degrees C and 43, 85 and 100% r.h. The number of E. coli O157:H7 on leaves (5.6 or 1 9 log CFU per leaf) inoculated using DW as a carrier medium increased significantly at 25 degrees C and 100% r.h. within 120 h but remained constant or decreased significantly under other test conditions. E. coli O157:H7 on leaves (5.4 log CFU per leaf) inoculated using PW as a carrier increased significantly within 72 and 24 h, respectively, at 12 or 25 degrees C and 100% r.h.; counts using a low inoculum (2.2 log CFU per leaf) increased significantly within 24 h at 25 degrees C. Conclusions: Escherichia coli O157: H7 can colonize on spinach leaves at 12 or 25 degrees C in a 100% r.h. environment. Organic matter in the inoculum carrier may provide protection and nutrients which enhance survival and colonization. Significance and Impact of the Study: Colonization of E. coli O157:H7 on spinach leaves as affected by organic matter in the inoculum, temperature and r.h. was determined. These observations will be useful when developing strategies to prevent growth of E. coli O157:H7 on pre- and postharvest spinach.