Methylxanthines, such as theophylline and caffeine, are widely used in premature neonates to prevent and treat apnea of prematurity. However, the effect of methylxanthines on the immature brain has not been examined in detail. To investigate the effects of methylxanthines on the immature brain, theophylline (Loading: 6mg/kg, Maintenance 2 or 6mg/kg every 12 hours) or caffeine (Loading: 10mg/kg, Maintenance 3 or 9mg/kg every 24 hours) was administered intraperitoneally to neonatal rats at P3 to P9 (7 days), and BrdU (50mg/kg) was injected at P9 and P10. Rats were sacrificed at P17. Both numbers of BrdU-positive cells and BrdU/NeuroD double positive cells in the dentate gyrus were decreased significantly in the rats treated with theophylline or caffeine, compared to the vehicle-treated rats. However, methylxanthines did not affect the ratio of BrdU/NeuroD double positive cells or BrdU/S100β double positive cells to total BrdU positive cells. In a subsequent experiment, neural stem/progenitor cells (NSPC) from rat fetuses on embryonic day 14 were cultured in the presence of theophylline (1μg/ml, 10μg/ml, or 100μg/ml) or caffeine (1μg/ml, 10μg/ml, or 100μg/ml). On day 6 of culture, growth of the NSPC was evaluated with a cell proliferation assay system (Premix WST-1), and the extent of cell death in cultures was estimated with an LDH-cytotoxic test kit. Both theophylline and caffeine suppressed growth of the NSPC in a dose-dependent manner. However, neither increased cell death. These results indicate that administration of methylxanthines to premature infants may induce suppression of neurogenesis.