We have examined the effect of a sandwich culture and a open-faced culture in a low oxygen atmosphere for the long-time culture of postnatal rat (P3) basal forebrain neurons in defined medium (B27/Neurobasal, Gibco). After 4 days in culture, although viability was better in the low oxygen (9% O 2 ) than in the normal oxygen (20% O 2 ) for cells plated at 2 10 5 /cm 2 , it was not significantly different for cells plated at 4 10 5 /cm 2 . The survival of sandwich cultured neurons plated at 2 10 5 /cm 2 was worse than that of open-faced cultured neurons after 4 days in culture. However, in the sandwich condition, substantial survival of neurons with a fine meshwork of neurites was evident after 25 days in culture, and AchE-positive neurons were observed in the presence of 100 ng/ml NGF. A large number of GFAP-positive cells was observed in the open-faced condition both in low oxygen and normal oxygen atmosphere after 11 days in culture. In contrast, very few GFAP-positive cells were found in the sandwich condition.It was reported that the 9% oxygen condition was not significantly different from the sandwich condition for the long-term culture of embryonic rat hippocampal neurons. Our data suggest that the 9% oxygen condition for postnatal rat neurons is not so effective as that for embryonic rat neurons. It may be because postnatal neurons require a large amount of oxygen for their survival. It was interesting that substantial survival was observed in the sandwich condition after 3 weeks in culture with near absence of glial proliferation. We have established a primary neural cell cuture technique from the postnatal rat CNS to study neurotrophic factors. However, it was necessary to culture neurons on a feeder layer of astroglial cells for the long-time culture. We conclude that a sandwich culture is a useful technique to study neurotrophic factors on postnatal rat CNS without any influence of factors from astroglial cells.