We isolated from the rat brain two cDNA clones encoding the zeta and theta subtypes of the 14-3-3 protein. Both clones encoded 245 amino acid sequences, which share a high sequence homology with each other and also with other subtypes of the 14-3-3 protein. The distribution of their mRNAs was determined in the developing brain, by in situ hybridization with subtype-specific oligonucleotide probes. At embryonic day 18, the zeta and theta subtype mRNAs were expressed at high levels throughout the brain and the spinal cord. Distribution patterns of the two mRNAs were distinct in the brain at postnatal day 21. The zeta subtype mRNA was distributed widely in the brain gray matter, and high levels of the transcripts were detected in various brain regions, including the neocortex, hippocampus, caudate-putamen, thalamus, cerebellar cortex, and several brainstem nuclei. On the other hand, high signal levels of the theta subtype mRNA in the gray matter were restricted to the cerebellar cortex and the hippocampus. In addition, significant signals for the theta subtype mRNA were found over the white matter, where cell bodies of glial cells are populated. The wide gene expression of the zeta and theta subtypes suggests their fundamental and essential role in the brain function, but the degrees of functional involvement by the respective subtypes would be heterogeneous between neuron and glia, and also among neuron types.