Spinal motor pools project to target muscles forming distinct rostrocaudal topographic maps during development and regeneration. To define the mechanisms underlying these neuromuscular maps we studied the preferential outgrowth of embryonic spinal cord neurites on muscle membranes from different axial positions and explored the role of ephrin A ligands. We found all five ephrin As (EphAs) expressed in serratus anterior, gluteus maximus and diaphragm muscles. In the diaphragm, four of the five ephrin As are expressed as a caudal to rostral gradient. When ephrin A function is disrupted in muscle membranes by deletion of glycosyl-phosphatidylinositol anchored ephrin A ligands with phosphatidylinositol-specific phospholipase C enzyme treatment or by blocking of ephrin A ligands with EphA fusion proteins, or by genetic manipulation leading to ephrin A2/A5 mutant mice, the spinal cord neurites loose their preference for the membranes of corresponding axial position; suggesting a significant role for ephrins in topographic choices made by growing motor neurons. To closely approximate topographic choices presented to embryonic neurites in vivo, neurites within the phrenic motor pool were challenged to make outgrowth choices on membranes of their normal target, the diaphragm muscle. We observed that neurites from rostral cervical segments (C1 and C2) prefer to grow on rostral diaphragm membranes; caudal cervical neurites (C6-C8) choose caudal diaphragm membranes; a transition of positional preference occurs at C4 and this ability is lost in ephrin A2/A5 mutant mice. These results demonstrate for the first time topographical outgrowth of axons from within a motor pool onto a single target muscle in vitro.