The purification, cloning, and deduced amino acid sequence of an ADP-ribosyltransferase secreted from the marine bacterium Vibrio fischeri (V. fischeri ADP-r) is described. This enzyme was purified from culture supernatant, and partial amino acid sequence obtained from the purified protein was used to design a degenerate oligonucleotide probe that was used to clone a cross-hybridizing DNA fragment from V. fischeri genomic DNA. Recombinant Escherichia coli clones harboring this fragment possessed ADP-ribosyltransferase activity. The DNA fragment was sequenced, and deletion analysis localized the ADP-ribosyltransferase activity to one of the three possible open reading frames in the fragment; the deduced amino acid sequence from this open reading frame matched the amino acid sequence obtained from the purified protein. V. fischeri ADP-r has no significant homology (DNA or amino acid) with other known ADP-ribosyltransferases. This enzyme appears to require neither proteolytic cleavage nor a reducing agent for enzymatic activity. The cloned gene is expressed but not secreted in E. coli; however, it is secreted from a heterologous marine Vibrio species. We have named this enzyme halovibrin.