A method for biological equilibration (BE) of allergen reference preparations using the skin-prick test (SPT) method and histamine HCl 10 mg/ml as reference substance (reference method), was evaluated. The precision was low for weals less than 10 mm2. The slope (log weal area/log concentration) of allergen and histamine did not vary significantly between investigators and allergens. The median slopes were 0.39 (n= 384) and 0.34 (n= 397), for allergen and histamine, respectively (P < 0.01). The concentration of allergen eliciting a weal of the same size as that of histamine HCl 1 mg/ml (Chl) in the median sensitive patient, 1000 Biological Units/ml (BU/ml), did not vary significantly between clinics/geographical regions (grasses, mites and moulds). As BE is repeatable between regions. BUs estimated by this method are generally valid. A high correlation (r= 0.91, P < 0.001) was found between the median Chl as estimated with histamine 1 and 10 mg/ml as reference substance, respectively. Thus, this reference method for BE is valid. The precision of the SPT method with histamine HCl 1 mg/ml is not as good as with 10 mg/ml, which is therefore recommended as the reference concentration.