The 17beta-hydroxysteroid dehydrogenase (17beta-HSD) enzymes are involved in the interconversion of biologically active and inactive sex steroids and are considered to play a critical role in the in situ metabolism of estrogen, especially in estrogen-dependent breast cancer. The gene encoding 17beta-HSD type 2 is located at 16q24.1-2, and earlier studies have shown that allelic loss in this region is an early and frequent event in breast cancer progression. Recurrence of hormone-dependent breast cancer frequently occurs several years after the primary treatment. The aim of this study was to investigate whether the expression of 17beta-HSD types 1 and 2 differs in tumors from patients with late relapses (>5 years) compared with controls without recurrence after long-term follow-up. Using real-time reverse transcription-PCR, we found that the normal mammary gland expressed both 17beta-HSD types 1 and 2, whereas the tumors frequently lacked detectable levels of type 2. Only 10% of the estrogen receptor-positive tumors expressed type 2, whereas 31% of the ER-negative tumors did so (P = 0.031). In a case-control series of 84 patients, a high level of 17beta-HSD type 1 indicated increased risk to develop late relapse of breast cancer (odds ratio, 3.0; 95% confidence interval, 1.0-12.6; P = 0.041), whereas retained expression of type 2 indicated decreased risk (odds ratio, 0.25; 95% confidence interval, 0.05-1.2; P = 0.050). In multivariate analysis of the estrogen receptor-positive patients, the absence of 17beta-HSD type 2 combined with a high expression of type 1 showed prognostic significance (P = 0.016) in addition to DNA aneuploidy (P = 0.0058), whereas progesterone receptor status did not (P = 0.71). These findings suggest that abnormal expression of 17beta-HSD isoforms has prognostic significance in breast cancer and that altered expression of these enzymes may have importance in breast cancer progression.