OBJECTIVE: The objective of the study was the estimation of the actual hospitalization costs of two neonatal intensive care units of university of Athens. METHODS: The study was based on retrospective data gathered from the records of 70 neonates who were hospitalized at the NICUs of Alexandra's obstetric and Aglaia Kyriakou pediatric hospitals of Athens, with admission dates between February and April 2004 until their discharge from the hospital. Clinical data derived from medical records, while economic data from each hospital's administrative and financial department. Regression analysis was performed with SPSS program in order to find the correlation between the cost and birthweight and other parameters. The mean daily treatment cost was estimated according to: birth weight and gestational age of the neonates as well as Length of Stay in NICU and Intermediate level II until their discharge from hospital. Direct cost analysis was based on cost of personnel, cost of supplies, medication, parenteral feeding, laboratory and imaging tests, cost of infrastructure, hotel services and various other on-site costs. The economic analysis did not include the depreciation of capital assets. The prices used for the analysis were based on Greek NHS prices, expressed in €2004. RESULTS: The mean daily actual cost per infant in the NICU of Alexandra was 207 euro and for intermediate level II €121. The mean daily actual cost per infant in NICU for Aglaia Kyriakou hospital was 511€ and for intermediate level II €231. The mean length of stay (LOS) in the NICU and Intermediate level II of Alexandra was 17,22 and 24,16 days respectively. The mean LOS in the NICU and Intermediate level II of A.Kyriakou was 8.5 and 11.5 days respectively. CONCLUSION: Estimates of the economic costs of preterm birth can be informative to decision-makers and facilitates quality improvement efforts used in neonatal care.