Previous studies have indicated that freely transferred osteomyocutaneous flaps may fail despite anastomotic patency. While microvascular dysfunction is thought to be one of the major causes for this type of flap failure, little is known of its underlying mechanisms, probably due to the lack of adequate experimental models allowing detailed intravital microcirculatory analysis. Herein we report quantitative analysis of the microcirculation of periosteum, muscle, subcutis and skin by intravital fluorescence microscopy using an osteomyocutaneous free flap model in the hindlimb of rats. The microcirculation of the different tissues was studied after microanastomotic transfer (free flap), and was compared to that after solely elevating the tissue, mimicking a pedicled osteomyocutaneous flap. Transferred flaps, which were exposed to 1 h of ischaemia during the anastomotic procedure, showed a slight but significant decrease (P< 0.05) of functional capillary density in muscle, subcutis and skin when compared with the microcirculation of pedicled flaps, while capillary diameters, red blood cell velocity and blood flow of perfused capillaries remained almost unaffected. The decrease of functional capillary density was associated by a significant (P< 0.05) inflammatory response, as indicated by the increased number of leukocytes adherent to the endothelial lining of postcapillary venules. While the functional capillary density of periosteum was not affected by the free transfer procedure, the inflammatory response was found similar when compared with that observed in muscle and subcutis. Thus, our study indicates that even after a short 1-h ischaemic time period, capillary perfusion failure and leukocyte-endothelial cell interaction are the main events, characterising microvascular dysfunction after free transfer of osteomyocutaneous flaps. Using the model described herein, intravital microscopic analysis of the microcirculation proved an appropriate tool to study the individual microvascular response after free tissue transfer, and may thus be used to evaluate the effectiveness of novel therapeutic regimens which aim at counteracting microcirculatory dysfunction in free osteomyocutaneous flaps. Copyright 1999 The British Association of Plastic Surgeons.