Axonin-1, a member of the immunoglobulin/fibronectin type-III family of cell-adhesion molecules, occurs both as a glycosylphosphatidylinositol-(glycosylPtdIns)-anchored membrane-bound and a soluble form. In vivo observations show that the major part of axonin-1 is found in the soluble fraction and that soluble axonin-1 perturbs neurite fasciculation and pathfinding in the developing chicken embryo. This has prompted further investigations into the mechanism of the axonin-1 release. We demonstrate here that axonin-1 released from dorsal root ganglion neurons contains ethanolamine and inositol, components of the glycosylPtdIns anchor. Secreted axonin-1 does not exhibit the cross-reacting determinant epitope, an indication that the cleavage of the anchor is not mediated by a phosphatidylinositol-specific phospholipase C. Treatment of dorsal root ganglion neurons with 1,10-phenanthroline, an inhibitor of glycosylPtdIns-specific phospholipase D, reduces the release of axonin-1 by 56%. Moreover, glycosylPtdIns-specific phospholipase D activity was detected in dorsal root ganglion neurons and brain. These results suggest that axonin-1 is released from the membrane by an endogenously expressed glycosylPtdIns-specific phospholipase D in vivo. With domain-swaping experiments between axonin-1 and its non-released relative F11, deletion mutants and monoclonal antibodies, we demonstrate that the fourth fibronectin type-III-like domain of axonin-1 is required for the generation of the soluble form of axonin-1.