OBJECTIVE To establish recommendations for prevention of vitamin K deficiency in healthy breastfed infants. METHODS Prospective clinical trials of different methods of vitamin K prophylaxis. METHODS Study of vitamin K1 and proteins induced by vitamin K absence (PIVKA)-II concentrations, and some coagulation factors at the ages of 2, 4, 8 and 12 weeks in healthy breastfed infants with either once 1 mg vitamin K1 orally (n = 165) or intramuscularly (n = 166), or weekly 1 mg orally (n = 48), or daily 25 micrograms orally (n = 58). RESULTS Despite significantly higher vitamin K1 plasma concentrations after intramuscular administration, there was no difference in activities of coagulation factors VII and X, and PIVKA-II concentrations between oral and intramuscular administration. The two single administrations of 1 mg could not prevent the appearance of PIVKA-II after the age of 1 month. When vitamin K was administered as 1 mg per week or 0.025 mg per day, significantly higher concentrations of vitamin K1 were found and no PIVKA-II was detectable. CONCLUSIONS A single administration of 1 mg vitamin K1 orally or intramuscularly may not afford complete protection against late vitamin K deficiency in healthy breastfed infants. A regimen of 1 mg per week or 25 micrograms vitamin K1 per day proved to be effective in prevention of vitamin K deficiency and the latter is recommended for breastfed infants during the first three months of life.