The domain structure of transcription termination factor rho was analyzed by partial trypsin cleavage and by photoaffinity labeling with ATP and oligo(C)5. A rho subunit consists of three distinct domains of nearly equal size that are connected by trypsin-sensitive linker segments. The amino-terminal domain binds to oligo(C)5 and has sequence segments with extended similarity to conserved elements in other RNA-binding proteins and a segment that has identities with another known cytidine nucleotide-binding protein. The middle domain has the ATP-binding site and has most of the sequence segments with similarity to conserved elements in other nucleoside triphosphate-binding proteins. The function of the third domain, the carboxyl-terminal domain, has not been identified. Both the amino- and carboxyl-terminal domains are relatively resistant to further trypsin treatment. The ATP-binding domain is also relatively resistant when it is linked to the amino-terminal, RNA-binding domain, but has not been detected as a separate digestion product. This result plus the finding that ADP and ATP inhibit the cleavage in the linker between the two domains indicate that those domains interact intimately in spite of their functional distinctions.