In the last decade the heme oxygenase (HO) system has been strongly highlighted for its potential significance in maintaining cellular homeostasis. Nevertheless the physiological relevance of the three isoforms cloned to date, HO-1, HO-2 and HO-3, and their reciprocal interrelation have been poorly understood. In the brain the HO system has been reported to be very active and its modulation seems to play a crucial role in the pathogenesis of neurodegenerative disorders. To discriminate the regional and cellular distribution of HO isoforms in the CNS, we have developed a real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) protocol. With this highly sensitive methodology we have assessed for the first time the expression of all known HO isoform mRNAs in different rat brain areas. Although they presented a highly dissimilar range of expression, with HO-2>HO-1>HO-3, all three HO isoform transcripts demonstrated high level of expression in the cerebellum and the hippocampus, showing in a different scale, a strikingly parallel distribution gradient. We have also quantified the expression of HO mRNAs in primary culture of cortical neurons and type I astrocytes. While HO-1 and HO-2 were detected in both cellular types, HO-3 transcript was uniquely found in astrocytes. To further investigate the regional brain expression of this elusive and poorly studied isoform, we have performed in situ hybridization using an HO-3 specific riboprobe. HO-3 mRNA was expressed mainly in hippocampus, cerebellum and cortex. The initial elucidation of HO isoforms distribution should facilitate further research on their pathophysiological role in the nervous system.