Progressive angiogenesis and endothelial differentiation in the chick chorioallantoic membrane (CAM) serve to accommodate oxygen demands of the growing embryo. The present study evaluated CAM microvascular endothelial permselectivity during the most rapid phase of angiogenesis (day 10) and after initiation of endothelial cytodifferentiation (day 14). Chick embryos were incubated using established shell-less culture techniques for intravital and ultrastructural observations. Systemic microinjections of FITC-dextrans (40, 70 and 150 KDa) provided an index of endothelial permselectivity after 2.5 min and 10 min perfusions. Ultrastructural examinations of the same dextran probes served to detect small, intermittent foci within the perivascular interstitium. Although minor variations of dextran particle distributions around specific segments of the microcirculation were observed ultrastructurally, perivascular accumulation was not sufficient to elicit a detectable fluorescent signal. Thus, substantial accumulation of the graded-dextran series in the perivascular interstitium was not detected. Morphometric analyses of the precapillary, capillary, and postcapillary microvascular segments served to demonstrate a continuous endothelium which displayed cytoplasmic attenuation at day 14. Plasmalemmal vesicles were few and uniform within the microvascular units at day 10. A three-fold increase in vesicle densities characterized the precapillary endothelia at day 14. Average widths of the endothelial junctional clefts were homogeneous within the segmental microvascular endothelia at both days 10 and 14. Junctional cleft lengths were also homogeneous, except the significantly longer capillary endothelial clefts observed at day 10. These results are consistent with the concept that, despite certain differences in segmental vesicle densities and junctional cleft lengths, neovascularization of the CAM is achieved without excessive macromolecular efflux across the microvascular endothelia.