The protein encoded by the UL14 gene of herpes simplex virus type 1 (HSV-1) and HSV-2 is expressed late in infection and is a minor component of the virion tegument. An UL14-deficient HSV-1 mutant (UL14D) forms small plaques and exhibits an extended growth cycle at low multiplicities of infection (MOI) compared to wild-type virus. Although UL14 is likely to be involved in the process of viral maturation and egress, its precise role in viral replication is still enigmatic. In this study, we found that immediate-early viral mRNA expression was decreased in UL14D-infected cells. Transient coexpression of UL14 and VP16 in the absence of infection stimulated the nuclear accumulation of both proteins. We intended to visualize the fate of VP16 released from the infected virion and constructed UL14-null (14D-VP16G) and rescued (14R-VP16G) viruses that expressed a VP16-green fluorescent protein (GFP) fusion protein. Synchronous high-multiplicity infection of the viruses was performed at 4 degrees C in the absence of de novo protein synthesis. We found that the presence of UL14 in the virion had an enhancing effect on the nuclear accumulation of VP16-GFP. The lack of UL14 did not significantly alter virus internalization but affected incoming capsid transport to the nuclear pore. These observations suggested that UL14 (i) enhanced VP16 nuclear localization at the immediately early phase, thus indirectly regulating the expression of immediate-early genes, and (ii) was associated with efficient nuclear targeting of capsids. The tegument protein UL14 could be part of the machinery that regulates HSV-1 replication.