Adenosine influences neurotransmitter release, neuronal excitability, and firing rate, through A1-adenosine receptors (A1-R). Caffeine and related methylxanthines are adenosine receptor antagonists. Exposure of developing rodents to caffeine is associated with subtle, long-term changes in neurochemistry and behavior. The developmental appearance of A1-R gene expression was examined in rats by in situ hybridization. On gestational day (GD) 10, A1-R mRNA was expressed at very high levels in placental mesometrium. Expression of A1-R mRNA in brain was first detected on GD 14. Hybridization was restricted to portions of neuroepithelium, caudate-putamen, piriform cortex, hypoglossal nucleus, and ventral horn of spinal cord. Neuroepithelial A1-R mRNA increased in intensity and distribution at subsequent ages, reaching a maximum on GD 20 (the latest age studied). Hybridization signal was detected, with regional variation in intensity, throughout much of the brain by GD 16, with additional increases in extent and intensity through GD 20. Generally, a caudal > rostral gradient of hybridization intensity was apparent. The distribution on GD 20 resembled the widespread yet heterogeneous pattern observed in the adult, with high levels of A1-R gene expression in cortex, hippocampus, thalamus, cerebellum, pontine nuclei, brainstem motor nuclei, and spinal cord. Northern blot analysis confirmed the age-related increase in abundance of A1-R transcripts (ca. 3.5 and 5.5 kb). The early and widespread expression of A1-R mRNA, coupled with previous reports of prenatal A1-R binding, suggests that adenosine and adenosine antagonists, including caffeine, may influence neuronal differentiation, migration or synaptogenesis, thus producing long-lasting effects on brain and behavior.