Two forms of clathrin light chains, LCa and LCb, are expressed in all mammalian and avian tissues that have been examined, whereas only one type is found in yeast. Regions of structural dissimilarity between LCa and LCb indicate possible functional diversity. To determine how LCa and LCb might differentially influence clathrin function, light chain expression patterns and turnover were investigated. Relative expression levels of the two light chains were determined in cells and tissues with and without a regulated secretory pathway. LCa/LCb ratios ranged from 5:1 to 0.33:1. A higher proportion of LCb was observed in cells and tissues that maintain a regulated pathway of secretion, suggesting a specialized role for the LCb light chain in this process. The ratio of light chains in assembled clathrin was found to reflect the levels of total light chains expressed in the cell, indicating no preferential incorporation into triskelions or coated vesicles. The half-lives of LCa, LCb, and clathrin heavy chain were determined to be 24, 45, and 50 h, respectively. Thus, LCa is turned over independently of the other subunits. However, the half-lives of all three subunits are sufficiently long to allow triskelions to undergo many rounds of endocytosis, minimizing the possibility that turnover contributes to regulation of clathrin function. Rather, differential levels of LCa and LCb expression may influence tissue specific clathrin regulation, as suggested by the predominance of LCb in cells maintaining a regulated secretory pathway.