After the anthrax incidents in October 2001, several techniques used for sampling surfaces for biological agents were found to be inadequately validated, especially at low surface loadings. Therefore a test chamber was developed to produce sample sets having targeted surface concentrations of dry biological agent simulant. Dry spore aerosols were initially dispersed into the chamber at relatively high air concentrations, and monitored in real time. The concentration decay (due to stirred settling and dilution) was measured and when the targeted air concentration was reached, the sampling surfaces were uncovered and exposed to the settling particles until >99% of the particles had settled. Multiple agar plates were used to estimate the true colony-forming-unit (CFU) surface concentration. The uniformity of surface loadings was limited by random deposition of small numbers of particles on the surfaces (Poisson distribution) and was characterized by how much greater the observed variability was than that predicted by Poisson statistics. The flow-enhanced powder mixture appeared to affect the spores' ability to grow on the agar medium. Three ways of analyzing the agar plates were used to evaluate the effect of spore coatings on viability and to differentiate between number of spore-containing particles and the number of spores. The presence of spore agglomerates re-suspended by various sample handling activities in the chamber further increased the variability of deposited particles. Based on estimated airborne particle concentration, it was possible to predict mean agar plate concentrations within narrow confidence intervals (CI) at low (4.8 CFU, 95% CI 3.5-6.4), medium (20 CFU, 95% CI 17-23), and high (160 CFU, 95% CI 140-190) concentrations.