The localization of nitric oxide synthases (NOS) is dealed with in the developing gonads of amphioxus Branchiostoma belcheri tsingtauense. It was found by NADPH-diaphorase staining that (1) NOS activity was present in the nuclear membranes of germinal vesicles during the entire period of oocyte development; (2) NOS was localized in both the nuclear membranes and the perinuclear region of cytoplasm in the vitellogenetic oocytes; (3) NOS was relocated in the cortical layer in the mature egg; (4) NOS activity was present in spermatocytes, but not in the spermatogonia in the middle of October; (5) NOS was detected in both spermatozoa and spermatids as well as spermatocytes during the breeding season. This is the first report on the distribution pattern of NOS in the developing gonads in protochordates. These results suggest a role for NOS in the functioning of the nuclear membranes and yolk synthesis during oogenesis and in cell division and differentiation during spermatogenesis.