Breast-feeding mothers frequently transmit cytomegalovirus (CMV) to preterm infants of very low birth weight. Current recommendations for prevention of virus transmission are based on data published 20 y ago in the context of human milk banking. Two recent clinical trials examined storage of breast milk at -20 degrees Celsius to reduce virus transmission. However, in both studies, CMV transmission occurred. Using sensitive tools like quantitative PCR, CMV pp67 late mRNA assay, and a high-speed, centrifugation-based microculture assay for quantification of CMV infectivity, we reassessed the virological and biochemical characteristics of freeze-storing breast milk at -20 degrees Celsius, compared it with traditional Holder pasteurization (30 min at 62.5 degrees Celsius), and a new short-term pasteurization (5 s at 72 degrees Celsius) based on the generation of a milk film. Both heat treatment procedures were able to destroy viral infectivity and pp67 RNA completely. Preliminary results showed short-term heat inactivation below 72 degrees Celsius was less harmful in reducing the activity of marker enzymes than Holder pasteurization. Freezing breast milk preserved the biochemical and immunologic quality of the milk; however, late viral RNA and viral infectivity was also preserved. Compared with viral DNA, CMV-RNA more directly reflects infectious CMV in human milk samples. Further studies are necessary to evaluate short-term heat treatment below 72 degrees Celsius as an effective tool for prevention of CMV transmission.