Nitric oxide (NO) is a ubiquitous, endogenously produced free radical, which is involved in a wide variety of biological processes, such as vasodilation, neurotransmission, blood clotting and host defence mechanisms (Ignarro, 1990; Moncada et al., 1991). NO is produced by conversion of oxygen and L-arginine to NO and L-citrulline. This reaction is catalysed by nitric oxide synthase (NOS), of which there are three different isoforms: endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) in almost all types of cell (Griffith and Stuehr, 1995). eNOS and nNOS are constitutive and Ca2+–calmodulin-dependent isoforms that are involved in cellular signalling. iNOS is an inducible isoform produced only in response to a stimulus and is not Ca2+ dependent. NO plays important roles in many events during embryonic development, such as regulation of egg activation at fertilization in sea urchin oocytes (Kuo et al., 2000), and regulation of the balance between cell proliferation and differentiation in Drosophila embryo development (Kuzin et al., 1996). In addition, several studies have reported the activities of the NO-related enzyme, NOS, in preimplantation development (Gouge et al., 1998) and implantation in mammals (Novaro et al., 1997; Biswas et al., 1998; Gagioti et al., 2000; Saxena et al., 2000). Although it has been reported that eNOS and iNOS are expressed in blastocysts collected from the uterus of delayed-implanting mice, and preovulatory and ovulatory oocytes (Jablonka-Shariff and Olson, 1997, 1998; Gouge et al., 1998), expression and activities of NOS in preimplantation embryos and the role of NO in preimplantation development has not been fully elucidated. In the present study, the presence and changes of NOS activities, and the distribution of eNOS and iNOS isoforms were examined in mouse embryos during preimplantation development and in unfertilized mouse oocytes at the second metaphase (MII) stage using NADPH-diaphorase histochemistry and immunofluorescence staining, respectively. Because NOS and NADPH diaphorase activities are induced by different properties of the same enzyme molecule, NADPH diaphorase activity can be used as a marker for NOS (Wang et al., 1995).