The developmental relations between abducens (VI) nerves and their targets, the lateral rectus, quadratus, and pyramidalis muscles, have been examined in the chick embryo from early neural tube stages through 10 days of incubation. Sites of myoblast origins were determined by microinjection of replication-incompetent retroviruses containing the LacZ reporter into paraxial mesoderm corresponding to somitomeres 3-5. Motor neurons and axons were identified by Bodian staining, immunocytochemistry, and application of DiI and DiO to dissected peripheral nerves. Anlage of the dorsal oblique originate in somitomere 3, close to the ventrolateral margin of the mid-to-caudal mesencephalon. Precursors of the lateral rectus arise deep within somitomere 4, beside the future metencephalon (rhombomere “A”). Quadratus and pyramidalis precursors are located between and partially segregated from these other two anlage. VIth nerve axons exit rhombomeres 5 and 6 via multiple median roots, fasciculate, and by stage 17 have elongated rostrally beneath the hindbrain. Immediately caudal to a mesenchymal pre-muscle condensation located deep to rhombomere 2, the VIth nerve separates into two branches. One branch enters the rostral portion of the condensation, from which quadratus and pyramidalis muscles will segregate. This branch projects exclusively from rhombomere 5 and is the accessory abducens nerve. The other branch enters the caudal, presumptive lateral rectus, region of the condensation. This is the abducens nerve, and it projects from cells located in both rhombomeres 5 and 6. These findings indicate that specific matching of motor nerves with their presumptive targets begins prior to the differentiation and segregation of myogenic populations, and that spatial organization of developing eye muscles is initiated well before they interact with connective tissue precursors derived from the neural crest. © 1994 Wiley-Liss, Inc.