Bovine papillomavirus type 1 (BPV-1) late gene expression is regulated at both transcriptional and posttranscriptional levels. Maturation of the capsid protein (L1) pre-mRNA requires a switch in 3′ splice site utilization. This switch involves activation of the nucleotide (nt) 3605 3′ splice site, which is utilized only in fully differentiated keratinocytes during late stages of the virus life cycle. Our previous studies of the mechanisms that regulate BPV-1 alternative splicing identified three cis-acting elements between these two splice sites. Two purine-rich exonic splicing enhancers, SE1 and SE2, are essential for preferential utilization of the nt 3225 3′ splice site at early stages of the virus life cycle. Another cis-acting element, exonic splicing suppressor 1 (ESS1), represses use of the nt 3225 3′ splice site. In the present study, we investigated the late-stage-specific nt 3605 3′ splice site and showed that it has suboptimal features characterized by a nonconsensus branch point sequence and a weak polypyrimidine track with interspersed purines. In vitro and in vivo experiments showed that utilization of the nt 3605 3′ splice site was not affected by SE2, which is intronically located with respect to the nt 3605 3′ splice site. The intronic location and sequence composition of SE2 are similar to those of the adenovirus IIIa repressor element, which has been shown to inhibit use of a downstream 3′ splice site. Further studies demonstrated that the nt 3605 3′ splice site is controlled by a novel exonic bipartite element consisting of an AC-rich exonic splicing enhancer (SE4) and an exonic splicing suppressor (ESS2) with a UGGU motif. Functionally, this newly identified bipartite element resembles the bipartite element composed of SE1 and ESS1. SE4 also functions on a heterologous 3′ splice site. In contrast, ESS2 functions as an exonic splicing suppressor only in a 3′-splice-site-specific and enhancer-specific manner. Our data indicate that BPV-1 splicing regulation is very complex and is likely to be controlled by multiple splicing factors during keratinocyte differentiation.