Recombinant Epstein-Barr viruses (EBV) with a translation termination codon mutation inserted into the nuclear protein 3A (EBNA-3A) or 3C (EBNA-3C) open reading frame were generated by second-site homologous recombination. These mutant viruses were used to infect primary B lymphocytes to assess the requirement of EBNA-3A or -3C for growth transformation. The frequency of obtaining transformants infected with a wild-type EBNA-3A recombinant EBV was 10 to 15%. In contrast, the frequency of obtaining transformants infected with a mutant EBNA-3A recombinant EBV was only 1.4% (9 mutants in 627 transformants analyzed). Transformants infected with mutant EBNA-3A recombinant virus could be obtained only by coinfection with another transformation-defective EBV which provided wild-type EBNA-3A in trans. Cells infected with mutant EBNA-3A recombinant virus lost the EBNA-3A mutation with expansion of the culture. The decreased frequency of recovery of the EBNA-3A mutation, the requirement for transformation-defective EBV coinfection, and the inability to maintain the EBNA-3A mutation indicate that EBNA-3A is essential or critical for lymphocyte growth transformation and that the EBNA-3A mutation has a partial dominant negative effect. Five transformants infected with mutant EBNA-3C recombinant virus EBV were also identified and expanded. All five also required wild-type EBNA-3C in trans. Serial passage of the mutant recombinant virus into primary B lymphocytes resulted in transformants only when wild-type EBNA-3C was provided in trans by coinfection with a transformation-defective EBV carrying a wild-type EBNA-3C gene. A secondary recombinant virus in which the mutated EBNA-3C gene was replaced by wild-type EBNA-3C was able to transform B lymphocytes. Thus, EBNA-3C is also essential or critical for primary B-lymphocyte growth transformation.