Multiple chitinases have been found in hyphae of filamentous fungi, which are presumed to have various functions during hyphal growth. Here it is reported, for the first time, the primary structure of one such intracellular chitinase, named chitinase III, from Rhizopus oligosporus, a zygomycete filamentous fungus. Chitinase III was purified to homogeneity from actively growing mycelia of R. oligosporus using three steps of column chromatography. Its molecular mass was 43.5 kDa and the pH optimum was 6.0 when p-nitrophenyl N,N',N"-beta-D-triacetylchitotrioside was used as a substrate. Chitinase III also hydrolysed chromogenic derivatives of chitobiose, but had no N-acetylglucosaminidase activity. The gene encoding chitinase III (chi3) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The deduced amino acid sequence of chi3 was similar to that of bacterial chitinases and chitinases from mycoparasitic fungi, such as Aphanocladium album and Trichoderma harzianum, but it had no potential secretory signal sequence in its amino terminus. Northern blot analysis showed that chi3 was transcribed during hyphal growth. These results suggest that chitinase III may function during morphogenesis in R. oligosporus.