The Epstein-Barr virus (EBV)-determined nuclear antigens EBNA 1, 2, 3, 4, and 6, regularly expressed in EBV-transformed lymphoblastoid cell lines, vary in size among viral strains. We have used this characteristic to trace the spread of the virus within seven families by using an approach called Ebnotyping. Among 33 evaluable individuals, 3 were EBV seronegative, and 17 different EBV strains could be isolated from the peripheral blood or throat washes of the remaining 30. All unrelated persons carried different strains. The EBV strain carried by 19 persons was also found in 1 or more family members. The same viral strain was carried by two members in five families, by three members in the sixth, and by five members in the seventh. The paternal strain was isolated from one child in two families, and the maternal strain was isolated from one or more children in three families. EBV was isolated from both blood and throat wash in six individuals. The Ebnotypes of both derived lymphoblastoid cell lines were identical within each individual. These results indicate that spread within families may be a relatively common route of EBV transmission. The number of horizontal transmission events required to generate diversification of the Ebnotype will require larger epidemiological studies.