In 2003, approximately 37% of pregnant women in Botswana (2001 population: 1.7 million; approximately 40,000 births per year) were infected with human immunodeficiency virus (HIV). Since 2001, all prenatal clinics in Botswana have offered HIV screening and interventions for prevention of mother-to-child transmission of HIV (PMTCT), which can decrease vertical transmission of HIV from 35%-40% to 5%-10%. Historically, HIV testing in Botswana has been performed after individual pretest counseling, with patients actively choosing whether to be tested (i.e., an "opt-in" approach). In 2003, 52% of pregnant women receiving prenatal care nationwide learned their HIV status. In 2004, to increase use of free national PMTCT and antiretroviral treatment (ARV) programs, Botswana began routine, noncompulsory (i.e., "opt-out") HIV screening in prenatal and other health-care settings. Concerns have been raised that routine testing in Africa might deter women from seeking prenatal care and might result in fewer women returning for their test results and HIV care after testing. To assess the early impact of routine testing on HIV-test acceptance and rates of return for care, the CDC Global AIDS Program and the PMTCT program in Botswana evaluated routine prenatal HIV testing at four clinics in Francistown, the second largest city in Botswana, where HIV prevalence has been > or =40% since 1995. This report describes the results of that assessment, which indicated that, during February-April 2004, the first 3 months of routine testing, 314 (90.5%) of 347 pregnant women were tested for HIV, compared with 381 (75.3%) of 506 women during October 2003-January 2004, the last 4 months of the opt-in testing period (p<0.001). However, many women who were tested never learned their HIV status because of logistical problems or not returning to the clinic. Substantial increases in HIV testing of pregnant women were also observed at the Francistown referral hospital and at prenatal clinics nationwide. These findings highlight the potential public health impact of routine HIV testing with rapid, same-day results for programs seeking to increase the number of persons with access to HIV-prevention and treatment services.