Retrospective chart review was undertaken for 480 patients who underwent a total of 506 valve replacements or repair procedures for infective endocarditis. The influence of preoperative antimicrobial treatment on culture, Gram stain, and histopathological examination findings for resected valve specimens was examined. When valves were removed before the end of treatment, organisms were seen on the Gram stain of ground valve material performed in the microbiology laboratory and on Gram-stained histopathological sections in 231 (81%) of 285 and 140 (67%) of 208 specimens, respectively (P=.0007). Gram-positive cocci were either cultured from or observed in excised valve tissue in 42 (67%) of 63 episodes involving negative preoperative blood cultures. Positive Gram stain results for microbiological specimens should be reintroduced into the definite pathological criteria for infective endocarditis. When deciding on how long to continue antimicrobial therapy after valve replacement for endocarditis, valve culture results should be the only laboratory finding taken into account, because it takes months for dead bacteria to be removed from sterile vegetations.