Expression of human papillomavirus late genes encoding L1 and L2 capsid proteins is restricted to terminally differentiated epithelial cells. We have previously identified and characterized an AU-rich, cis-acting negative regulatory element in the 3' untranslated region of human papillomavirus type 1 late mRNAs. This element acts posttranscriptionally to reduce mRNA levels and the translation efficiency of mRNAs. The experiments reported here are a continuation of our previous work. We have used RNA gel shifts and UV cross-linking assays to identify cellular proteins that interact with the inhibitory RNA sequence of human papillomavirus type 1. RNA gel shift assays established that cellular proteins interact with the AU-rich sequence. The binding of nuclear proteins was inhibited by competition with poly(U), whereas the binding of cytoplasmic proteins was inhibited by competition with poly(U) and also by competition with poly(A) and poly(G). Two nuclear proteins and two cytoplasmic proteins that bind specifically to the AU-rich RNA sequence were identified by UV cross-linking. These proteins did not bind to the 3' untranslated region of human papillomavirus type 1 early mRNAs, which does not show inhibitory activity. The cellular proteins identified in our experiments may therefore be involved in the inhibition of human papillomavirus type 1 late gene expression in nondifferentiated epithelial cells.