A fluorescent substance was detected in elastin and in an alkali-soluble, hydroxyproline-free protein (ALSP) extracted from elastic tissue during the purification procedure for elastin. This fluorescent substance had ion exchange and paper chromatographic properties identical to those of dityrosine, the o,o'-biphenol analog of tyrosine, which has been proposed to be and interchain crosslink in the rubber-like insect protein, resilin. Radioscopic experiments indicated its derivation from tyrosine. This fluorescent compound was isolated in pure form from ALSP from adult bovine ligamentum nuchae. A comparison of both the gel permeation, ion exchange and paper chromatographic properties, as well as the ultraviolet absorption and fluorescence characteristics of the isolated compound with those of synthetic dityrosine confirmed its identity as dityrosine. Both in the case of bovine ligamentum nuchae and day-old chick aorta, dityrosine was present in greater quantities in ALSP as compared to elastin; dityrosine could not be detected in elastin from adult bovine ligamentum nuchae. Solubility, gel permeation, and ion exchange characteristics of ALSP indicated that, like similar glycoproteins reported by others from a variety of connective tissues, ALSP exists in various states of aggregation. The dityrosine content of ALSP was found to be directly related to the size, extent of aggregation and insolubility of the protein. Introduction of additional dityrosine into ALSP by treatment with horseradish peroxidase was accompanied by an increase in aggregation and a decrease in solubility of the protein. After extensive enzymic hydrolysis of ALSP, dityrosine was concentrated in the enzyme resistant portion of the protein. On the basis of these observations, dityrosine was proposed to be an interchain crosslink in ALSP. Other crosslinks may also be present in ALSP, some of which may be derived from dityrosine. Studies of the ammino acid compositions of aortic elastin from fetal and young chicks of various ages indicated that alkali-purified elastin was associated with a considerable amount of a more polar protein. The amino acid composition of this polar, alkali-insoluble contaminant appeared to be similar to that of ALSP. Moreover, this polar contamination was most persistent in the youngest of tissues. The morphology of developing elastic fibres and the persistence of the polar contamination suggested that ALSP may be involved in the biosynthesis of elastin. A hypothesis for this role is presented.