A primary culture of cortical neurons was established from a 17-day-old rat embryo. Twenty-four hours after plating (day 0), the medium was changed to a chemically defined one. Hypoxia was exerted for 4 h by placing cultures in an air-tight chamber connected to a vacuum pump and a cylinder filled with 95% N2 and 5% CO2. Hypoxic stress was exerted on one of the consecutive days from day 0 to day 3. Hypoxia on days 0, 1 or 2 reduced the number of surviving neurons immediately following exposure. However, hypoxia on day 3 resulted in delayed reduction. Anit-synaptophysin antibody was used immunocytochemically to examine whether the difference is related to teh synaptic formation of cultured neurons. Neurons which had more and longer neurites were clearly stained for synaptophysin. We suspect that neurons with synaptic formation release substances from their presynaptic vesicles, and that these substances might account for the delay reduction.