Assay methods for varicella-zoster virus (VZV) susceptibility to acyclovir (ACV) of VZV were compared by using cell-free (CF) and cell-associated (CA) virus of 6 x plaque-purified VZV. The 50% effective doses (ED50) of ACV, as required to reduce virus plaque formation by 50%, were about 8 times higher for CA virus than for CF virus. Also, the ED50 of 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU) for CA-VZV was higher than for CF-VZV, and fresh clinical isolates of VZV gave higher ACV ED50 values than CF virus. CA virus prepared at various times after CF virus infection showed a gradual increase of the ACV ED50 with time, ranging from the ED50 for CF virus to that for CA virus.