The sensitivity of microbiological assay methods for sulfonamides in muscle, kidney, serum, and milk was improved with Standard II Nähragar (Merck 7883), the medium being supplemented with trimethoprim, 1% sodium chloride, and 0.4% dextrose. The agar was seeded with 10(4) spores of Bacillus subtilis BGA per ml medium. The trimethoprim concentrations per ml agar were, at pH 6.20, pH 7.0, and pH 8.0 respectively 0.30, 0.15 and 0.07 microgram. The sulfonamide sensitivity of the test method depended on the pK-value of the drug tested and on the pH of the agar. For sulfonamides tested in muscle homogenate the optimum sensitivity of the bioassay methods was in the range of 0.32-0.63 microgram/ml; in kidney homogenate it was 0.32-0.63 microgram/ml; in serum 0.32-0.63 microgram/ml; and in milk 0.08-0.32 microgram/ml. The sensitivity for dapsone ranged in these substrates from 0.01 to 0.02 microgram/ml. The assay methods here developed may be used for both qualitative detection and quantitative determinations of sulfonamides in animal tissues, serum, and milk.