We investigated the antimicrobial activity of four polymyxin B components, B1, B2, B3, and isoleucine (Ile)-B1, individually and in combination. B3 was the most active agent against all organisms tested except Acinetobacter baumannii, for which Ile-B1 was most active. One combination met the criteria for synergy, B3 plus Ile-B1. No combinations exhibited antagonism. The dominant components of polymyxin B products (B1 and B2) were associated with the lowest probability of improved antibacterial activity when combined.