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A distinctive feature of human IgG4 is its ability to recombine half molecules (H chain and attached L chain) through a dynamic process termed Fab-arm exchange, which results in bispecific Abs. It is becoming evident that the process of Fab-arm exchange is conserved in several mammalian species, and thereby represents a mechanism that impacts humoral immunity more generally than previously thought. In humans, Fab-arm exchange has been attributed to the IgG4 core-hinge sequence (226-CPSCP-230) in combination with unknown determinants in the third constant H chain domain (CH3). In this study, we investigated the role of the CH3 domain in the mechanism of Fab-arm exchange, and thus identified amino acid position 409 as the critical CH3 determinant in human IgG, with R409 resulting in exchange and K409 resulting in stable IgG. Interestingly, studies with IgG from various species showed that Fab-arm exchange could not be assigned to a common CH3 domain amino acid motif. Accordingly, in rhesus monkeys (Macaca mulatta), aa 405 was identified as the CH3 determinant responsible (in combination with 226-CPACP-230). Using native mass spectrometry, we demonstrated that the ability to exchange Fab-arms correlated with the CH3-CH3 dissociation constant. Species-specific adaptations in the CH3 domain thus enable Fab-arm exchange by affecting the inter-CH3 domain interaction strength. The redistribution of Ag-binding domains between molecules may constitute a general immunological and evolutionary advantage. The current insights impact our view of humoral immunity and should furthermore be considered in the design and evaluation of Ab-based studies and therapeutics. [ABSTRACT FROM AUTHOR]