Boundaries in the Bithorax complex (BX-C) of delimit autonomous regulatory domains that drive parasegment-specific expression of homeotic genes. BX-C boundaries have two crucial functions: they must block crosstalk between adjacent regulatory domains and at the same time facilitate boundary bypass. The C2H2 zinc-finger protein Pita binds to several BX-C boundaries, including and To study Pita functions, we have used a boundary replacement strategy by substituting modified DNAs for the boundary, which is located between the and regulatory domains. Multimerized Pita sites block crosstalk but fail to support regulation of (bypass). In the case of , we used a novel sensitized background to show that the two Pita-binding sites contribute to its boundary function. Although is from BX-C, it does not function appropriately when substituted for : it blocks crosstalk but does not support bypass. Mutation of the Pita site disrupts blocking activity and also eliminates dCTCF binding. In contrast, mutation of the dCTCF site does not affect Pita binding, and this mutant boundary retains partial function.