We evaluated Hybrid Capture 2 (HC2) and polymerase chain reaction (PCR) results for paired specimens collected at 19,187 visits from 5,026 of 5,060 women participating in the Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesion Triage Study (ALTS). We examined the test agreement between HC2 and PCR detection for any of 13 carcinogenic human papillomavirus types targeted by HC2 and compared clinical performance of the 2 tests for detecting concurrent and follow-up cervical intraepithelial neoplasia (CIN) 3 or cancer. The k value for the 2 assays was 0.65 (95% confidence interval, 0.64-0.66), with 82.7% crude agreement. HC2 was more sensitive (93.6% vs 89.3%; P < .0005) but less specific (41.2% vs 48.5%; P < .0005) than PCR for detecting 2-year cumulative CIN 3 or cancer (n = 503). The presence of multiple types as detected by PCR and/or cytologic abnormality increased the likelihood of an HC2+ result. Increased sensitivity of HC2 compared with PCR was surprising, given the theoretical advantages of PCR-based methods for analytic sensitivity. Smaller amounts of material used in PCR could have limited its sensitivity, but our results demonstrate the importance of optimization and standardization of PCR-based assays for clinical applications.