The UL24 gene of herpes simplex virus type 1 is conserved across many herpesviruses, but its protein product has not been identified. We expressed the UL24 gene in insect cells from a recombinant baculovirus and used the resulting protein to raise a rat antiserum. In immunoblotting experiments, this antiserum recognized a 30-kDa protein in lysates from infected cells. The identity of this species as UL24 was confirmed by using a virus encoding a truncated form of UL24. On the basis of biochemical fractionation of infected cells, UL24 appeared to be predominantly nucleus associated, especially at later times in infection. Although certain UL24 transcripts exhibit early kinetics, UL24 protein accumulated at later times in infection and levels were decreased sixfold in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid; thus, UL24 was expressed with leaky-late kinetics. The entire UL24 open reading frame is encoded by mRNAs with two different 5′ ends. A mutation that eliminates the more abundant transcripts that originate at the first transcription start site resulted in a 10-fold reduction in the level of UL24 expressed but did not eliminate expression. Thus, the less abundant transcripts originating at the second transcription start site can evidently be translated, although transcripts originating at the first start site appear to be the major contributors to the expression of UL24. We conclude that UL24 is a bona fide herpes simplex virus type 1 protein that associates primarily with nuclei and whose expression is regulated at multiple levels.