The U(s)11 gene of herpes simplex virus 1 maps in the unique sequences of the short component of the HSV-1(F) genome approximately 775 bp from the center of the DNA replication origin (Ori(s)) and encodes a virion protein which binds RNA in sequence- and conformation-specific fashion, negatively regulates the accumulation of a prematurely terminated transcript of U(L)34, associates in the infected cell with the 60S ribosomal subunit, and, late in infection, accumulates in nucleoli. We report the following: (i) Deletion of a 222-bp sequence including Ori(s) (ΔOri(s)) negatively affected the accumulation of the U(s)11 protein without decreasing the accumulation of the U(s)11 transcript. (ii) The defect, observed at all times after infection, was multiplicity independent, was unrelated to U(s)11 protein stability, and apparently resulted from a cis-acting element since a coinfecting virus was unable to complement the ΔOri(s) virus. (iii) Transcription from the U(s)11 promoter initiated from three sites on the ΔOri(s) virus. Transcripts initiated from two of the three initation sites accumulated similarly in cells infected with the ΔOri(s) virus or wild-type parent virus. The low- abundance transcript initiating from the third site was apparently unique to the ΔOri(s) virus but was not expected to alter the coding capacity of the mRNA. (iv) Infected cells accumulated RNA derived by antisense transcription of the genome domain containing the U(s)11 gene. One transcript accumulated in larger amounts in cells infected with the ΔOri(s) virus than in cells infected with parent or repaired virus.