HPV‐16 viral load has been assessed with real‐time PCR assays by measuring HPV‐16 DNA and a human gene in genital samples. HPV‐16 viral load measurements are thus based on the inference that inhibitors contained in samples will equally impede amplification of DNA sequences from HPV‐16 and human DNA. We have previously shown that sample lysates can inhibit amplification of HPV‐16 but not β‐globin DNA. In the current study, cervicovaginal lavages lysates considered adequate for PCR analysis by a qualitative β‐globin PCR test, were screened for the presence of inhibitors using internal controls (IC) for HPV‐16 DNA and β‐globin in real‐time PCR assays. Of 150 lysates screened with both ICs, 12 (8%) contained inhibitors. Inhibition of amplification of both ICs was demonstrated in four of these specimens. In eight lysates, amplification of HPV‐16 IC only was impeded. Six (50%) of these 12 lysates tested positive for HPV‐16 DNA despite the presence of PCR inhibitors. The HPV‐16 viral load increased significantly after dilution of 11 of 12 lysates, demonstrating the presence of inhibitors in the undiluted lysate. Nine (90%) of 10 samples with inhibitors that were tested after dilution did not demonstrate inhibitory activity. The use of internal controls in real‐time PCR is clearly essential to determine HPV viral loads since the effect of inhibitors on primer‐driven genomic amplification is variable. J. Med. Virol. 72:132–137, 2004. © 2004 Wiley‐Liss, Inc.