SUMMARY Arginine in the medium was essential for the replication of herpes simplex virus in RK I3 cells. Virus adsorption, penetration and eclipse were not affected. When the medium of arginine-deficient infected cultures was exchanged for complete medium, virus growth resumed with the appearance of a near-synchronous infection. Resumption of virus growth was immediate if the exchange was made in the first 7 days, but if it was later a delay resulted which increased as the duration of deprivation increased. A threshold concentration of arginine was found for herpes virus growth. Virus yield was increased when the arginine concentration was raised over a limited range, but further addition of arginine did not continue to improve the yield. Undialysed calf serum could not be substituted successfully for arginine m herpes virus replication, although it supported cell growth and the replication of vaccinia and coxsackie B 3 viruses.