One of the challenges facing the development of programs to reduce mother to child transmission of HIV in developing countries remains the problem of infant feeding. One of the alternative feeding methods under investigation for infants of HIV-infected mothers is heat treatment of expressed breastmilk by Pretoria Pasteurization. The objective of this study was to determine the effect of Pretoria Pasteurization on commensal and pathogenic bacteria in hand-expressed human breastmilk, and to determine the duration of time for which milk can be kept safely without refrigeration after Pretoria Pasteurization. Samples of milk were hand expressed by lactating women in the postnatal ward. The samples were split into control and pasteurized specimens. The pasteurized specimens underwent Pretoria Pasteurization. All samples were stored at room temperature and were sampled for bacterial culture every 4 h, up to 12 h. Clinically significant levels of bacterial contamination occurred in 59 per cent of control and 7.8 per cent of pasteurized samples. Four pasteurized samples showed significant contamination. There is strong evidence that the contaminating organisms in these samples were introduced by handling after pasteurization. The 53 (91 per cent) pasteurized samples that had no contamination at 4 h remained sterile for the remainder of the standing period of 12 h. Forty-one per cent of control samples already had significant growth after standing at room temperature for 4 h. In conclusion, Pretoria Pasteurization kills pathogenic and commensal bacteria in hand-expressed breastmilk. Expressed breastmilk that has undergone Pretoria Pasteurization can be kept without refrigeration for up to 12 h with minimal probability of bacterial contamination provided that it is kept in the pasteurization container and is not handled.