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Using immunofluorescence technique we have analysed the Rb, p53, EBNA-2 and EBNA-5 expression pattern in EBV infected human B-cells and established lymphoblastoid cell lines (LCL-s). Resting B-cells showed only a faint Rb and no p53 immunostaining. The expression of both Rb and p53 increased after EBV infection. The change was first detectable 6 h after infection. The frequency of brilliantly Rb positive cells increased more rapidly than p53 positives. EBNA-2 and EBNA-5 became first detectable 12 h after infection. The frequency of EBNA positive cells in the freshly infected cultures was concordant with the proportion of CD23 and PCNA positives, but remained consistently below the frequency of Rb and p53 positive cells. Double immunofluorescence staining showed that all EBNA-5 positive cells were strongly Rb and p53 positive. LCL-s did not stain for p53, whereas the Rb staining was maintained at a high level. The EBNA-5 staining pattern changed from brilliant almost homogeneous nuclear staining in the freshly infected B-cells, to a nonhomogeneous pattern with a small number of strongly fluorescent nuclear bodies in established LCL-s. There was no change in the EBNA-2 staining pattern. Our findings indicate that the immortalization of B-cells by EBV may initially involve a high expression of EBNA-5, p53 and Rb, but only cells with low p53 and focal expression of EBNA-5 in nuclear bodies have the selective advantage required to grow into immortalized lines.