Collagen fibril assembly is a multistep process involving multiple macromolecular interactions. Type XIV collagen contains multiple domains and is capable of interacting with collagen fibrils and other extracellular matrix components. During tendon development, naturally changing expression of type XIV collagen and its variants may modulate such interactions. Type XIV collagen was studied using immunochemical and molecular approaches. Western analysis demonstrated that type XIV collagen content was high between days 14 and 19, decreasing sharply at hatching. Immunoelectron microscopy demonstrated that type XIV collagen was fibril-associated, with a periodicity of 67 nm, indicating specific interactions. Decreased fibrilassociated reactivity for type XIV collagen was seen at hatching, indicating a removal of collagen XIV from the fibril surface. The expression of two NC1 splice variants was analyzed. Overall, type XIV collagen mRNA decreased significantly from day 14 to hatching. The long NC1 splice variant was the predominant species at 14 days; at 19 days the two variants were expressed in lower amounts at nearly a 1:1 ratio; at hatching, both variants were expressed minimally. Changes in splice variant expression, suggest that different functional forms of type XIV collagen are present, allowing modified interactions with fibrils during development. In conclusion, type XIV collagen is fibril-associated and developmentally regulated. Modulation of expression of the NC1 splice variants may mediate the fibril interactions that allow the transition from growing fibril intermediates to mature fibrils.