Cells cultured from laryngeal papillomas contain episomal human papillomavirus type 6 or type 11 (HPV-6/11) DNA. We developed a sensitive RNase protection assay to simultaneously measure expression from the HPV E6, E7, and E1 promoters (P1, P2 and P3, respectively) in this manipulable culture system and found that P1, P2 and P3 transcript abundances could be independently modulated by culture medium composition and culture substrate. In undifferentiated cells grown in a low-calcium, serum-free medium, P1 transcripts commonly predominated over those from P2, P3 transcripts were often undetectable, and high concentrations of retinoic acid were able to selectively decrease P2 transcript abundance. When cultures were allowed to stratify and differentiate by growth on a collagen gel at he air-liquid interface, total HPV RNA increased up to sixfold because of selective increases in abundances of P1 and P3 transcripts. High-calcium submerged cultures also showed easily detectable P3 transcripts, and isolated suprabasal cells contained almost exclusively these transcripts. Growth arrest alone was not sufficient to induce P3 transcripts. Thus, in contrast to the HPV-6/11 E6 and E7 promoters, the E1 promoter was utilized primarily in a differentiation-specific manner. We also show that increased HPV gene dosage will not necessary bring about increased HPV transcript abundance, suggesting that other viral and cellular factors are responsible for regulation of total transcript levels as well as specific promoter usage.