The aim of this investigation was to assess the immunological properties of several insulin analogues by examining both their antigenic and immunogenic behaviour. In addition, the hypoglycaemic activity was also determined and compared with values obtained with natural insulin. The modified insulin preparations were of porcine and bovine origin. All analogues had in common the fact that the alanine B30 had been exchanged by either leucine, threonine, tyrosine, phenylalanine or glycine. The blood glucose-lowering activity was determined in rabbits and dogs, while the stimulation of antibody development was studied in three different animal species; pigs, dogs and rabbits. The antigenic properties of analogues were evaluated in vitro by measuring their binding capacity to pre-formed antibodies. In all cases the blood glucose lowering activity of the analogues was comparable to that of the respective natural insulin. There were remarkable differences in the binding capacity to pre-formed antibodies, with bovine Leu-B30 insulin competing only to 73% with the natural insulin tracer. With regard to antibody development, the analogues behaved similarly to the original hormones. These results show that there is little correlation between the antigenic make-up of the insulin molecule and its ability to provoke antibody stimulation.