Although the Centers for Disease Control and Prevention (CDC) recommend routine HIV counseling, testing, and referral (HIVCTR) in settings with at least a 1 percent prevalence of HIV, roughly 280,000 Americans are unaware of their human immunodeficiency virus (HIV) infection. The effect of expanded screening for HIV is unknown in the era of effective antiretroviral therapy. We developed a computer simulation model of HIV screening and treatment to compare routine, voluntary HIVCTR with current practice in three target populations: "high-risk" (3.0 percent prevalence of undiagnosed HIV infection; 1.2 percent annual incidence); "CDC threshold" (1.0 percent and 0.12 percent, respectively); and "U.S. general" (0.1 percent and 0.01 percent). Input data were derived from clinical trials and observational cohorts. Outcomes included quality-adjusted survival, cost, and cost-effectiveness. In the high-risk population, the addition of one-time screening for HIV antibodies with an enzyme-linked immunosorbent assay (ELISA) to current practice was associated with earlier diagnosis of HIV (mean CD4 cell count at diagnosis, 210 vs. 154 per cubic millimeter). One-time screening also improved average survival time among HIV-infected patients (quality-adjusted survival, 220.7 months vs. 219.8 months). The incremental cost-effectiveness was 36,000 dollars per quality-adjusted life-year gained. Testing every five years cost 50,000 dollars per quality-adjusted life-year gained, and testing every three years cost 63,000 dollars per quality-adjusted life-year gained. In the CDC threshold population, the cost-effectiveness ratio for one-time screening with ELISA was 38,000 dollars per quality-adjusted life-year gained, whereas testing every five years cost 71,000 dollars per quality-adjusted life-year gained, and testing every three years cost 85,000 dollars per quality-adjusted life-year gained. In the U.S. general population, one-time screening cost 113,000 dollars per quality-adjusted life-year gained. In all but the lowest-risk populations, routine, voluntary screening for HIV once every three to five years is justified on both clinical and cost-effectiveness grounds. One-time screening in the general population may also be cost-effective. Copyright 2005 Massachusetts Medical Society.