Herpes simplex virus type 1 capsids bud at nuclear and Golgi membranes for envelopment by phospholipid bilayers. In the absence of U(S)3, nuclear membranes form multiple folds harboring virions that suggests disturbance in membrane turnover. Therefore, we investigated phospholipid metabolism in cells infected with the U(S)3 deletion mutant R7041(ΔU(S)3), and quantified membranes involved in viral envelopment. We report that (i) [(3)H]-choline incorporation into nuclear membranes and cytoplasmic membranes was enhanced peaking at 12 or 20 h post inoculation with wild type HSV-1 and R7041(ΔU(S)3), respectively, (ii) the surface area of nuclear membranes increased until 24 h of R7041(ΔU(S)3) infection forming folds that equaled ~45% of the nuclear surface, (iii) the surface area of viral envelopes between nuclear membranes equaled ~2400 R7041(ΔU(S)3) virions per cell, and (iv) during R7041(ΔU(S)3) infection, the Golgi complex expanded dramatically. The data indicate that U(S)3 plays a significant role in regulation of membrane biosynthesis.