There has been considerable recent interest in the development of the circulation in the zebrafish. Optical techniques typically used to visualize changes in heart size allow measurement of stroke volume during early vertebrate development, but this approach is complicated in zebrafish larvae because of the heart's irregular shape and its significant change in morphology during the first 6 d of development. By use of a three-dimensional integration of the early zebrafish heart and vessels, we have greatly reduced measurement error of stroke volume and cardiac output and have determined the cross-sectional growth of major vessels in the developing zebrafish larvae. A dramatic 500%-600% increase in cardiac output (from 10 to 50-60 nL min(-1)) occurs on days 5 and 6 postfertilization in Danio rerio. Cross-sectional area of key vessels (dorsal artery, caudal artery, dorsal vein) as well as between-individual variation significantly decreased over the first 6 d of development. Associated with the decrease in cross-sectional area is a significant increase in red blood cell velocity on days 5 and 6 postfertilization. Together, the three-dimensional data of the cardiac and vascular systems have shown that the most profound physiological and developmental changes occur in days 5 and 6, which corresponds with the appearance of the adult form of the heart and the transition from diffusive to convective O-2 supply to internal tissues.