The enzyme diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) pyrophosphohydrolase has been purified to homogeneity from firefly lanterns. It is a single polypeptide of M(r) 16,000 with a Km for Ap4A of 1.9 microM and kcat = 3.6 s-1. It is inhibited competitively by adenosine 5'-tetraphosphate (Ki = 7.5 nM) and non-competitively by fluoride ions (Ki = 50 microM). The specific activity of the enzyme in crude extracts of at least 20 milliunits/mg protein is 10-100 times higher than in any other eukaryote so far examined. Interestingly, firefly luciferase is known to synthesize Ap4A and related adenine-containing dinucleoside tetraphosphates in vitro. The high activity of Ap4A hydrolase in lanterns may be related to this ability and could be relevant to the use of the luciferase gene as a reporter gene.