Heme oxygenase (HO)-2, the constitutive cognate of oxidative stress inducible HO-1 (HSP32), degrades heme to biliverdin, carbon monoxide, and iron. The highest levels of HO-2 are found in the testis. Previously we identified multiple HO-2 homologous transcripts that differ in size and use three different 5' UTRs that form the untranslated first exon of the gene (referred to as rHO-2, rHO-2-1 and rHO-2-2) and two poly(A) signals. Also, we have characterized a functional glucocorticoid response element (GRE) in the promoter region of rHO-2. In this study, we have examined the structural basis for size heterogeneity of HO-2 transcripts and whether expression of HO-2 at mRNA and protein levels is subject to regulation by corticosterone. Age and tissue-dependence of transcript expression were examined as well. Our data indicate that the remarkable increase in HO-2 mRNA in adult rat testis is due primarily to generation of two HO-2 homologous transcripts of approx. 2.1kb and approx. 1.45kb size that use rHO-2 and are unique to this tissue, and that rHO-2 is not used within other organs. These transcripts are not present in the brain, kidney, thymus, heart, spleen, liver, or in prepubertal 14day old rat testis. The testis-specific transcripts contain all of the coding region exons present in the approx. 1.3kb and approx. 1.9kb transcripts that are common to all organs, including the adult and prepubertal rat testis. Differential use of the poly(A) signals accounts for the difference in size of these two transcripts. Treatment of newborn rats with corticosterone for 5days, starting on day 2 after birth, induced HO-2 protein expression in the testis as detected by Western blotting. In adult rat testis, corticosterone treatment, however, was not an effective regulator of HO-2 transcript populations or levels. The findings suggest that HO-2 levels in the testis are controlled by glucocorticoids; and that developmental and tissue-specific factor(s) determine generation of transcripts unique to the organ. The apparent exclusive use of rHO-2 by the mature testis is consistent with the possibility that HO-2 may play a role in male reproduction.