Glycinergic neurons are the major inhibitory neurons in the vertebrate central nervous system. In teleosts, they play important roles in the escape response by regulating the activity of the Mauthner (M-) cells. Here we studied the contact between glycinergic axons and the M-cells in early zebrafish embryos by double immunostaining with an anti-glycine antibody and the 3A10 antibody that labels M-cells. We also studied a transgenic line, Tg(GlyT2:GFP), in which GFP is expressed under the control of the promoter for the glycine transporter-2 gene. The initial contacts by ascending glycinergic axons on the M-soma were observed within 27h post-fertilization (hpf) on the lateral part of the ventral surface of the M-soma. Stochastic labeling of glycinergic neurons was then performed by injecting a GlyT2:GFP construct into early cleaving eggs. We identified the origin of the earliest glycinergic axons that contact the M-soma as commissural neurons, located in the anterior spinal cord, whose axons ascend along the lateral longitudinal fascicles with a short descending branch. We also found, in the fourth rhombomere, late-developed glycinergic commissural neurons whose axons contact anterior or posterior edge of both M-somas. This study provides the first example of the initial development of an inhibitory network on an identifiable neuron in vertebrates.