Gamma phage specifically lyses vegetative cells of Bacillus anthracis and serves as part of the basis for identification of isolates from agar cultures. We report our study to standardize gamma phage production and preparation and to validate the assay for routine use. Unstable phage preparations were largely reduced through propagation of phage on blood agar cultures of the avirulent B. anthracis strain CDC684 and were adequately stable for extended storage beyond 1 to 2 years at 4 degrees C, provided that the preparation initially gave rise to clearly discernible plaques (macroplaques, 5 to 10 mm in diameter) on dilution at 1:8 or greater during potency testing with the Sterne strain or its equivalent. The primary intent of the assay was to test nonhemolytic, ground-glass-appearing bacterial B. anthracis-like colonies arising from culture of clinical or nonclinical samples on 5% sheep blood agar. Specifically, the assay was designed to show clear or primarily clear circular zones of lysis on bacterial lawns at the site of gamma phage inoculation after incubation at 35 degrees C +/- 2 degrees C for 20 h. When tested with 51 B. anthracis strains and 49 similar non-B. anthracis Bacillus species, the analytical specificity was >95%, a value that is intentionally low because our study design included two rare nonsusceptible B. anthracis strains as well as a rare susceptible non-B. anthracis strain, B. cereus ATCC 4342. Repeatability, day-to-day precision, and analyst-to-analyst precision were superior. The assay was rugged to variations among phage lots, phage concentration, amounts of bacterial inoculum, and incubation times as short as 6 to 8 h. System suitability evaluation showed improved robustness when bacterial lawns were tested with high- and low-density inoculum using the first and second quadrants of a serial four-quadrant streak on 5% sheep blood agar plates.