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Background: There has been debate in the role of exogenous testosterone as a risk factor for stroke. Hormone replacement therapy (HRT) is considered a risk factor for stroke. The risk of ischemic stroke may increase when using testosterone-containing HRT.Methods: Using data from the observational component of the Women’s Health Initiative (WHI) [WHI Observational Study (OS)], we analyzed the 93,676 women aged 50-79 years, who participated in the OS over a period of 12±1 years. We compared the outcome of stroke in participants with reported use of a combination of testosterone and estrogen, estrogen alone, progesterone alone, and a combination of estrogen and progesterone, as recorded at the baseline visit. A logistic regression analysis was run to determine the odds of developing stroke.Results: Of the 93, 676 participants, 1772 used a combination of testosterone and estrogen (Estratest) HRT, 11,282 used progesterone alone, 10,808 used a combination of estrogen and progesterone, and 31,673 used estrogen alone. A smaller proportion of participants who developed an outcome of stroke had used Estratest as compared to estrogen alone or a combination of estrogen and progesterone (1.9% vs. 96.3% p=0.62). In the logistic regression, participants who had used Estratest were 1.2 times as likely to develop stroke as users of other hormone replacement therapy (OR 1.2 95%CI (0.96-1.6)), while women who had used progesterone only were 0.87 times less likely to develop stroke than users of other hormone replacement therapy (OR 0.874 95%CI (0.77-0.99)). After adjusting for confounders, the risk of developing stroke increased in users of Estratest (OR 1.25 95%CI (0.96-1.6) p=0.04), and decreased in users of progesterone only (OR 0.873 95%CI (0.77-0.99) p=0.038).Conclusion: Use of testosterone-containing HRT slightly increased the risk of stroke in women when compared to progesterone alone HRT, although this was not found to be significant. Stroke risk with Estratest may be considered to be similar to estrogen only and combination of estrogen plus progesterone HRT. Future studies are required to investigate these correlations.