The thymidine kinase produced in cell cultures by several orthopoxviruses has been tested for sensitivity to inhibition by thymidine triphosphate (TTP). Thymidine kinase preparations from eleven variola isolates and from five "whitepox" isolates were all found to be sensitive to inhibition by TTP. Preparations from other orthopoxviruses, including vaccinia, cowpox, and monkeypox, were all resistant to inhibition by TTP. There was no overlap between the two groups of results and no viruses were found which gave intermediate results. Tests with hybrid viruses showed that this character segregated independently of other biological marker characters and is thus a valuable additional marker for variola and variola-related viruses.