Dopamine (DA) and norepinephrine (NE) derivatives play an important role in the sclerotization and pigmentation of insect cuticles by serving as precursors for cuticular cross-linking. Protein preparations from prepupae of the medfly, Ceratitis capitata, were able to conjugate β-alanine with DA producing N-β-alanyldopamine (NBAD) or with NE, synthesizing N-β-alanylnorepinephrine (NBANE). The latter reaction has been demonstrated for the first time. Apparent kinetic parameters were obtained for both substrates, DA (V m a x =30.7+/-6.0pmolmin - 1 mg - 1 ; K m =29.5+/-3.5μM) and NE (V m a x =16.1+/-6.6pmolmin - 1 mg - 1 ; K m =89.0+/-8.3μM). The same protein seems to be responsible for both enzymatic activities, judging from several criteria like identical behavior under heat inactivation as well as identical Mg 2 + and Mn 2 + dependent stimulation and Co 2 + inhibition. Furthermore, the melanic mutants niger of C. capitata and ebony 4 of D. melanogaster, known to be defective for NBAD synthase, were also unable to synthesize NBANE. The protein preparation acylated tyrosine with much less efficiency, to produce sarcophagine (β-alanyltyrosine). Strikingly, extracts from the melanic mutants were unable to synthesize sarcophagine. Our results strongly suggest that the enzymatic activity previously known as NBAD synthase is in fact a novel catalytic protein showing broad substrate specificity. We propose to identify it as catecholamine-β-alanyl ligase.