The question of whether timing is involved in generating the topographic organization of the earliest embryonic projection from the retina to the tectum has been examined in Xenopus laevis. First, the normal schedule of axonal outgrowth from the retina to the tectum was characterized. Groups of axons originating from either dorsal or ventral extremes of the retina were labeled by in vitro incubation of sectors (one-quarter to one-third) of eye primordia in [3H]proline and their time courses of outgrowth were determined using light microscope autoradiography. Comparisons of the growth of dorsal and ventral nerve fiber populations showed that those from the dorsal retina leave the eye first, grow along the optic pathway, and reach the tectum roughly 6 hr ahead of those from the ventral retina. This stereotyped sequence of outgrowth schedules the development of the initial retinotectal map: first the ventrolateral tectum receives input from the dorsal retina (stage 37/38), and then the dorsomedial part receives input from ventral retina (stage 40). Second, to test whether the accurate timing of axon outgrowth and target invasion defines the spatial ordering of the earliest connections, the normal schedule of retinal fiber outgrowth was altered by substituting dorsal halves of young stage 21 eye primordia, labeled with [3H]proline, for those in older stage 27 embryos. These heterochronic transplants resulted in retarded outgrowth from the dorsal retina such that the original pioneer fibers reached the tectum at least 9 hr later than normal, arriving after ventral retinal fibers, thereby reversing the normal sequence of tectal invasion. Despite this, the initial pattern of tectal innervation remained spatially normal. It is concluded that the retinotectal map is generated not by the temporal sequence of retinal axon outgrowth but, rather, by selective means of neuronal recognition.