A considerable subset of oropharyngeal squamous cell carcinomas (SCCs) are positive for human papillomavirus (HPV); however, delineating etiologically-associated HPV infections from SCCs with concurrent HPV infection unrelated to tumorigenesis is challenging. Viral load assessment in biopsy specimens may help facilitate such differentiation. HPV16 viral load and serologic markers were assessed among oral and oropharyngeal cases from a multinational study conducted by the International Agency for Research on Cancer (IARC). HPV16 viral load, measured semiquantitatively by PCR-enzyme immunoassay, was dichotomized as high or low based on the median optical density value. Serologic antibodies to HPV16 virus-like particles (VLPs) and to HPV16 E6 and E7 proteins were measured by ELISA. Compared to HPV DNA-negative cases (n = 852), HPV16 DNA-positive cases with high viral load (n = 26) were significantly more likely to originate in the oropharynx (odds ratio [OR], 12.0; 95% confidence interval [CI], 5.2–27.5) and, after adjustment for tumor site (AdjOR), have antibodies against HPV16 VLPs (AdjOR, 14.6; 95% CI, 6.0–35.6), E6 (AdjOR, 57.6; 95% CI, 21.4–155.3) and E7 (AdjOR, 25.6; 95% CI, 9.3–70.8). HPV16 DNA-positive cases with low viral load (n = 27) were more commonly oropharyngeal (OR, 2.7; 95% CI, 1.1–6.2) and seropositive for HPV16 VLPs (AdjOR, 2.7; 95% CI, 1.1–6.9), E6 (AdjOR, 3.0; 95% CI, 0.7–14.0) and E7 (AdjOR, 3.5; 95% CI, 0.7–16.3), compared to HPV DNA-negative cases; the associations, however, were neither as strong nor as significant as the associations for high viral load. As there appears to be a strong association between HPV16 serologic markers and viral load, in the absence of data on serologic markers, HPV16 viral load may be used to help delineate the subset of HPV16 DNA-positive oral and oropharyngeal cancers that may be the consequence of HPV infection. © 2005 Wiley-Liss, Inc.