Development of antibody to pre-early nuclear antigen (anti-PENA) in persons with primary cytomegalovirus (CMV) infection was tested in serial serum specimens of four renal transplant patients and four patients undergoing open heart surgery using the anticomplement-immunofluorescent test (ACIF). In patients undergoing open heart surgery seroconversion of anti-PENA was mostly concomitant with the rise of IgG or IgM antibodies determined in the indirect or direct enzyme-linked immunosorbent assay (ELISA) whereas in all renal transplant patients developing anti-PENA a delayed rise of this antibody compared to IgG and IgM antibodies was observed. Significant rise of anti-PENA accompanied by an increase of IgG and IgM antibodies in indirect and direct ELISA was also found in three patients undergoing open heart surgery with recurrent CMV-infection. Anti-PENA was shown to persist longer than IgM antibody. Moreover, anti-PENA was present in the serum of nearly two-thirds of 30 persons with IgG antibody but without IgM antibody. It is concluded that antibody determination to PENA can serve as an additional means of diagnosing primary and recurrent CMV infections. Because of its long persistence only seroconversion or significant rise of this antibody may be considered evidence of infection. In some patients a delayed development of anti-PENA must be taken into consideration.