First observed in 1999, seed rot has now been reported in all cotton producing counties in South Carolina. Microbial isolations made from both symptomatic and asymptomatic bolls have revealed a diverse microbial community present in healthy and symptomatic cotton boll tissue. Commonly isolated microorganisms include members of the following genera, Enterobacter, Pseudomonas, Bacillus, Pantoea, and Cedecea. Twenty-four, single-colony purified strains isolated from symptomatic boll tissue were screened under greenhouse conditions for their ability to induce the seed rot symptoms observed under field conditions. Sixteen flower or soil-inoculated isolates induced at least twice the level of seed rot as observed for water-treated controls. Twelve seed-rot-positive isolates tested in the greenhouse, along with 29 additional isolates, were screened in microplot studies in the field. Nine isolates that induced seed rot under greenhouse conditions were unable to do so under field microplot conditions. Two isolates, Enterobacter agglomerans 33 and Cedecea neteri 86 induced seed rot symptoms under both greenhouse and microplot conditions at twice, or greater than, the levels observed in the water-treated controls. The involvement of a microbial agent in the occurrence of seed rot will be discussed.