We used a computer-based competency assessment tool for Gram stain interpretation to assess the performance of 278 laboratory staff from 40 laboratories on 40 multiple-choice questions. We report test reliability, mean scores, median, item difficulty, discrimination, and analysis of the highest- and lowest-scoring questions. The questions were reliable (KR-20 coefficient, 0.80). Overall mean score was 88% (range, 63%-98%). When categorized by cell type, the means were host cells, 93%; other cells (eg, yeast), 92%; gram-positive, 90%; and gram-negative, 88%. When categorized by type of interpretation, the means were other (eg, underdecolorization), 92%; identify by structure (eg, bacterial morphologic features), 91%; and identify by name (eg, genus and species), 87%. Of the 6 highest-scoring questions (mean scores, > or = 99%) 5 were identify by structure and 1 was identify by name. Of the 6 lowest-scoring questions (mean scores, < 75%) 5 were gram-negative and 1 was host cells. By type of interpretation, 2 were identify by structure and 4 were identify by name. Computer-based Gram stain competency assessment examinations are reliable. Our analysis helps laboratories identify areas for continuing education in Gram stain interpretation and will direct future revisions of the tests.