We have analyzed the role of fumonisins in infection of maize (Zea mays) by Gibberella moniliformis (anamorph Fusarium verticillioides) in field tests in Illinois and Iowa, United States. Fumonisin-nonproducing mutants were obtained by disrupting FUM1 (previouslyFUM5), the gene encoding a polyketide synthase required for fumonisin biosynthesis. Maize ear rot, ear infection, and fumonisin contamination were assessed by silk-channel injection in 1999 and 2000 and also by spray application onto maize silks, injection into maize stalks, and application with maize seeds at planting in 1999. Ear rot was evaluated by visual assessment of whole ears and by calculating percentage of symptomatic kernels by weight. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of applied strains in kernels was determined by analysis of recovered isolates for genetic markers and fumonisin production. Two independent fumonisin-nonproducing (fum1-3 and fum1-4) mutants were similar to their respective fumonisin-producing (FUM1-1) progenitor strains in ability to cause ear rot following silk-channel injection and also were similar in ability to infect maize ears following application by all four methods tested. This evidence confirms that fumonisins are not required for G. moniliformis to cause maize ear rot and ear infection.