Two fixation methods based on formaldehyde or acetone for qualitative cytomegalovirus antigenemia assay were evaluated on 405 consecutive blood samples. Cytomegalovirus was detected in 40 samples by the antigenemia assay: 36 were detected by formaldehyde fixation; 22, by acetone; and 18, by both methods. Differences were statistically significant (P = 0.0043). In addition, four fixation methods (two based on formalin [with and without permeabilization] and two using acetone at different fixation times) for quantitative antigenemia assay in a different set of 32 samples from known viremic patients were evaluated. Formalin-based methods were superior to acetone-based methods, showing statistically significant differences in either the number of positive samples detected (P < 0.02; McNemar test) or the mean positive cell counts (P < 0.003; two-tailed Student's t test for paired samples). No differences between the two formalin-based methods were found. We recommend the formaldehyde fixation procedure without subsequent permeabilization because of its simplicity and sensitivity.