Alberta, Canada, uses an "opt-out" strategy for prenatal HIV testing. We examined demographic characteristics of and HIV seroprevalence among women opting out. The proportion of total specimens that were from women who opted out decreased from 4.3% in 2002 to 3.6% in 2004 (P<.001), and HIV seroprevalence among these specimens was 0.07%, which was 3.3 times higher than the HIV seroprevalence among specimens from women who opted in. Women opting out may have a higher HIV infection risk.