SummaryA study was conducted to evaluate the biological activity ofBrucellaallergens extracted with hydrochloride or trichloroacetic acid. Smooth and mucoidBrucella abortuscells and the medium in which brucellae were propagated were used to prepare the allergens. The biological activity of the allergens was estimated in guinea‐pigs sensitized withBrucella abortusstrain 544. The guinea‐pigs were intradermally injected with several allergen dilutions. The dilutions were coded and randomized for site of injection so that none of the dilution was injected twice on the same site. Variance analysis using incomplete Latin square was used for the statistical calculation of the results. The calculated biological activity of the allergens was compared with the biological activity of a ‘standard’ allergen that has proved effective in detecting cattle brucellosis.The skin erythema diameter was best when recorded 32 h after allergen injection. Statistical analysis of the skin erythema diameters showed a great variation in biological activity (12–105%) between the allergens. Only the allergen extracted from the medium in which a mucoidBrucellastrain was propagated was as potent as the standard. The use of the incomplete Latin square for variance analysis resulted in the estimation of the biological activity of nine batches of allergen in only 27 gu