This work was initiated to address the gaps identified by Congress regarding validated biothreat environmental sampling and processing methods. Nine Laboratory Response Network-affiliated laboratories participated in a validation study of a cellulose sponge wipe-processing protocol for the recovery, detection, and quantification of viable Bacillus anthracis Sterne spores from steel surfaces. Steel coupons (645.16 cm(2)) were inoculated with 1 to 4 log(10) spores and then sampled with cellulose sponges (Sponge-Stick; 3M, St. Paul, MN). Surrogate dust and background organisms were added to the sponges to mimic environmental conditions. Labs processed the sponges according to the provided protocol. Sensitivity, specificity, and mean percent recovery (%R), between-lab variability, within-lab variability, and total percent coefficient of variation were calculated. The mean %R (standard error) of spores from the surface was 32.4 (4.4), 24.4 (2.8), and 30.1 (2.3) for the 1-, 2-, and 4-log(10) inoculum levels, respectively. Sensitivities for colony counts were 84.1%, 100%, and 100% for the 1-, 2-, and 4-log(10) inocula, respectively. These data help to characterize the variability of the processing method and thereby enhance confidence in the interpretation of the results of environmental sampling conducted during a B. anthracis contamination investigation.