The periorbital sheath serves as a major pathway for sympathetic nerves traveling to distal orbital targets in the rat. This tissue accommodates sympathetic fiber sprouting in the neonate but becomes impassable by postnatal day 30 (PND 30). In contrast, smooth muscle target remains receptive to sympathetic ingrowth. To determine the attributes of receptive and nonreceptive tissues, we compared periorbital pathway and target tissue phenotypes prior to (PND 5 and PND 15) and after (PND 30 and PND 60) the period when pathway receptivity is lost. Both pathway cells and superior tarsal smooth muscle cells expressed alpha-smooth muscle actin and smooth muscle myosin heavy chain throughout development. At PND 5-15, both tissues also expressed vimentin, collagen IV, laminin 1 and laminin beta2, whereas fibronectin was detected only in pathway tissue. At PND 30, vimentin, collagen IV, and fibronectin were absent in tarsal muscle but were robust in pathway tissue. Laminin 1 and laminin beta2 expression was maintained in muscle; however, in pathway cells, laminin 1 declined modestly, and laminin beta2 decreased precipitously to barely detectable levels. Quantitative competitive polymerase chain reaction showed that nerve growth factor mRNA was present in the pathway throughout development at levels that were greater than both surrounding connective tissue and tarsal muscle. We conclude that the loss of pathway receptivity to sympathetic nerve ingrowth is associated with a transition from a phenotype similar to fetal smooth muscle cells to one that is more consistent with myofibroblast-like cells.