Eruption of the third and fourth mandibular premolars in the dog begins at 15 weeks of age, is dependent upon the dental follicle, and is complete by 23 weeks. Our study covered the period from 12 to 20 weeks, and revealed several changes in extracellular matrix structure and organization of the follicle which correlate with specific physiological events in eruption. First, the average DNA content per follicle reached a maximum at 14 weeks. Two weeks later, follicle size had increased 1.3- to 2.4- times. Second, the collagen content of follicles increased 2.5-fold over the study period, with two-thirds of this increase over the last four weeks. Type I collagen was the major collagen at all stages of follicular development. The amount of proteoglycan rose 45% from 16 to 20 weeks of age. Third, the ultrastructure of the dental follicle prior to eruption (12 weeks) indicated a disorganized interstitial connective tissue matrix; during eruption, two size classes of fibrils were observed which clustered together in linearly aligned bundles. Fourth, gel electrophoretic analyses resolved more than twenty follicle proteins with the major species a M_r = 95 k glycoprotein. Immunoblotting demonstrated only one minor component was derived from serum. Comparison of noncollagenous proteins from different aged follicles indicated that three small polypeptides (M_r = 20–25 k) were present primarily at 16 weeks, the same time at which root elongation begins. A different sequence of changes was exhibited by two other proteins of M_r = 13 and 15 k. These findings may serve as biochemical markers of stages of dental follicle development and facilitate a search for local control mechanisms.