The structural integrity of herpes simplex virus 2 (HSV-2) during freezing, thawing, and lyophilization has been studied using scanning and transmission electron microscopy. Viral particles should be thawed quickly from -80 to 37 &DEG; C to avoid artifacts of thawing. To avoid freezing damage, the virus should be rapidly frozen (> 20 K s(-1)) rather than slowly frozen as occurs on the shelf of a lyophilizer (< 1 K s(-1)). Fast freezing and thawing allows six cycles of freeze thaw with no loss of viral titer TCID50. Viral particles were characterized using immunogold labeling methods. Freshly thawed virus had 19 &PLUSMN; 4 polyclonal immunogold particles virus(-1); virus stored at -80 &DEG; C for at least 4 months had 17 &PLUSMN; 3 particles virus-1; virus stored for 1 week at 4 &DEG; C had 8 &PLUSMN; 4 particles virus(-1). By bulk lyophilization the number of particles was 4 &PLUSMN; 4, but by fast freezing and lyophilization the number of gold particles improved to 12 &PLUSMN; 5. The loss of viral membrane was directly observed, and the in vitro loss was demonstrated to occur through three possible pathways, including (i) simultaneous release of tegument and membrane, (ii) sequential release of membrane and then tegument, and (iii) release like by in vivo infection. The capsids were not further degraded as indicated by the lack of free DNA, which was only released by boiling the viral samples with 1% SDS, followed by a dilution to 0.001% w/v SDS for the real-time PCR reaction.