In the present study rabbits were immunized with insulin preparations of various degrees of purity. Antibody formation was determined by Boyden's haemagglutination method (Boyden 1951), by Berson et al.'s chromatoelectrophoresis (Berson et al. 1956) and by agar electrophoresis In the two last mentioned methods highly purified 131I-pig insulin was used. The agglutination method gave a positive reaction with sera from rabbits immunized with the two least purified preparations, A-insulin and B-insulin, thus suggesting the formation of antibodies against impurities in these two preparations. On the other hand, agar electrophoresis, and to a lesser degree chromatoelectrophoresis, gave positive reactions with sera from all groups. Thus it was shown that antibodies were also formed in rabbits immunized with even the most purified insulin preparation. Antibody formation was, however, most pronounced in those rabbits which had been vaccinated with the most impure insulin preparation. By continuous paper electrophoresis the purest of the insulin preparations used for immunization could be separated into two fractions. The slowest migrating fraction tested by the haemagglutination test showed a weak but positive reaction indicating traces of impurities. Thus, although the possibility of a cross reactivity between insulin and the impurities demonstrated cannot be rejected, the results obtained do not allow of any definite decision on this question. The negative inhibition with the purest insulin preparation, however, suggests that there is no common antigenicity.