The presence or absence of the Es-Si esterase (female specific sex-influenced esterase) in rat sera was reexamined by the zymogram technique using agarosegel electrophoresis. It was found that female rat sera of the BN/Kyo strain, which has been typed as the Es-Si esterase negative, display an esterase migrating between the Es-1A esterase and the Es-2C esterase. Using a specific alloantibody to the esterase, it was shown that this esterase is identical to the Es-Si esterase. Thus, BN rats are the first described inbred rats possessing the Es-Si esterase, as well as the Es-1A esterase. Mating experiments between BN rats and SHR rats confirmed a previous demonstration that the presence or absence of the Es-Si esterase is controlled by a single gene locus closely linked to the Es-2 locus where the Es-1 locus also is linked. A further survey with 39 strains by the present methods revealed that besides two sublines of the BN strain, E3/Han, LOU/Max and PVG/Max strains also possess both the ES-1A esterase and the Es-Si esterase. The results indicate that these esterases are controlled by separate gene loci.