The EBV-determined nuclear antigen (EBNA) was studied with regard to several histochemical properties. Proteolytic enzymes destroyed EBNA staining. RNAse DNAse and hyaluronidase had no effect on the number of EBNA positive cells. Intensive treatment with DNAse weakened the chromosomal fluorescence of EBNA, whereas the staining of interphase nuclei was relatively enzyme resistant. When EBV-associated soluble complement-fixing antigen of Raji cells (CFA-R) was added to methanolacetic acid-fixed chicken red blood cells, brilliant and specific EBNA staining was obtained by anti-complement fluorescence (ACIF) with anti-EBNA positive sera. DNAse treatment abolished the ability of the nuclei to bind the antigen (CFA-R), whereas DNAse treatment following CFA-R antigen binding had no effect on the ACIF staining. EBNA was relatively heat stable. Somewhat weakened, but still fully positive EBNA reaction was obtained after 56 degrees C heating for 30 minutes in BSS. Periodate destroyed the EBNA reaction. Formaldehyde also abolished the staining, whereas methanol, acetone, methanol-acetone and methanol-hexyleneglycol-water preserved the staining relatively well.