The southeastern United States has an increasing burden of HIV, particularly among blacks, women, and men who have sex with men. To evaluate HIV nucleic acid amplification testing (NAAT) and antibody-based algorithms in determination of HIV incidence, detection of acute HIV infections, and surveillance of drug-resistant virus transmission in the urban southeastern United States, we conducted a cross-sectional analysis of prospectively collected data from 2202 adults receiving HIV testing and counseling at 3 sites in Atlanta, GA from October 2002 through January 2004. After standard testing with an HIV enzyme immunoassay (EIA) and Western blot confirmation, HIV-positive specimens were tested with 2 standardized assays to detect recent infection. HIV antibody-negative specimens were pooled and screened for HIV using NAAT. Seventy (3.2%) of 2202 subjects were HIV infected. Only 66 were positive on the standard HIV antibody test; 4 were antibody-negative but acutely HIV infected. The overall annual HIV incidence was 1.1% (95% confidence interval [CI]: 0.4 to 1.8) based on the Vironostika-LS assay and 1.3% (95% CI: 0.6 to 2.1) based on the BED Incidence Enzyme Immunoassay (EIA). The prevalence of acute HIV infection was 1.8 per 1000 persons (95% CI: 0.7 to 4.6). The sensitivity of the current testing algorithm using an EIA and Western blot test for detectable infections was only 94.3% (95% CI: 86.2 to 97.8). All 3 of the acutely infected subjects genotyped had drug resistance mutations, and 1 had multiclass resistance. Adding NAAT-based screening to standard HIV antibody testing increased case identification by 6% and uncovered the first evidence of multidrug-resistant HIV transmission in Atlanta. Antibody tests alone are insufficient for public health practice in high-risk urban HIV testing settings.