Human TK- cells carrying the HSV-2 TK gene as a result of transformation with virus DNA express a TK activity of virus origin and maintain the TK+ phenotype when grown in HAT medium. Under non-selective conditions, however, reversion to a TK- phenotype occurs with a significant frequency characteristic of each transformed line. Once reversion has occurred the TK- phenotype appears to be stable, since only very rare instances of TK- to TK+ reversion have been observed. TK- revertants were susceptible to re-transformation by virus DNA, but no reactivation of a silent virus TK gene could be obtained by superinfecting them with a TK- virus mutant. The data presented are consistent with the hypothesis that acquisition of the TK- phenotype is brought about by loss of the virus sequences coding for TK.