As the prevalence of heterosexually transmitted HIV increases among women of childbearing age in the United States, so too does the potential for vertical transmission from mother to child. Early maternal diagnosis and appropriate management are critical to minimizing the risk of perinatal infection. We designed a study to evaluate current prenatal care provider testing practices and knowledge of HIV as it relates to pregnancy in a low seroprevalence state. A written questionnaire was mailed to 642 prenatal care providers in Kentucky. Responses were compared to a similar survey conducted in 1998 and to current federal guidelines for HIV management. Nearly all respondents reported to offer HIV testing to all prenatal patients, demonstrating a marked improvement since 1998 (p < 0.001). However, clinicians did not report adequate follow-up when testing is refused and appear to have limited knowledge of the disease as it relates to pregnancy. Only 9.3% of respondents demonstrated proficiency on two knowledge assessment questions. Those with previous experience treating prenatal patients with HIV were more likely to respond correctly (odds ratio [OR] 3.03; 95% confidence interval [CI] 1.08-8.50). Providers with little experience treating patients with HIV may not possess the basic knowledge required to manage the disease during pregnancy. Additional educational interventions are needed in low seroprevalence areas to ensure the appropriate treatment of all HIV-positive pregnant patients and to minimize the risk of preventable perinatal transmission.