By ultracentrifugation of 30 ml of highly dilute suspensions of herpes simplex virus (HSV) directly onto monolayer cultures grown in centrifuge tubes, infectivity was significantly greater than without centrifugation. Ultracentrifugation at 20,000 to 25,000 rpm (28,000 to 45,000 X g) for 1.5 to 2.3 h was utilized with good preservation of cultures. With low-speed centrifugation at 3,000 rpm (1,100 X g), infectivity was almost 10-fold greater than without centrifugation. With ultracentrifugal inoculation, infectivity was about 100-fold greater than without centrifugation. Ultracentrifugal inoculation permitted the detection of HSV at concentrations as low as 0.05 plaque-forming units per ml. Similarly, ultracentrifugal inoculation of cultures was almost 100-fold more sensitive a method of detecting infectious HSV than was pelleting HSV from dilute suspensions followed by resuspension and inoculation of cultures. Ultracentrifugal inoculation of cultures may permit the isolation of HSV in situations where virus cannot be detected by ordinary means and may prove applicable to the study of other viruses.