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In Africa, prevention of mother-to-child HIV transmission (PMTCT) programs are hindered by limited uptake by women and their male partners. Routine HIV counseling and testing (HCT) during labor has been proposed as a way to increase PMTCT uptake, but little data exist on the impact of such intervention in a programmatic context in Africa.In May 2004, PMTCT services were established in the antenatal clinic (ANC) of a 200-bed hospital in rural Uganda; in December 2004, ANC PMTCT services became opt-out, and routine opt-out intrapartum HCT was established in the maternity ward. We compared acceptability, feasibility, and uptake of maternity and ANC PMTCT services between December 2004 and September 2005.HCT acceptance was 97% (3591/3741) among women and 97% (104/107) among accompanying men in the ANC and 86% (522/605) among women and 98% (176/180) among their male partners in the maternity. Thirty-four women were found to be HIV seropositive through intrapartum testing, representing an 12% (34/278) increase in HIV infection detection. Of these, 14 received their result and nevirapine before delivery. The percentage of women discharged from the maternity ward with documented HIV status increased from 39% (480/1235) to 88% (1395/1594) over the period. Only 2.8% undocumented women had their male partners tested in the ANC in contrast to 25% in the maternity ward. Of all male partners who presented to either unit, only 48% (51/107) came together and were counseled with their wife in the ANC, as compared with 72% (130/180) in the maternity ward. Couples counseled together represented 2.8% of all persons tested in the ANC, as compared with 37% of all persons tested in the maternity ward.Intrapartum HCT may be an acceptable and feasible way to increase individual and couple participation in PMTCT interventions.