Muskelin is an intracellular protein with a C-terminal kelch-repeat domain that was initially characterized as having functional involvement in cell spreading on the extracellular matrix glycoprotein thrombospondin-1. As one approach to understanding the functional properties of muskelin, we have combined bioinformatic and biochemical studies. Through analysis of a new dataset of eight animal muskelins, we showed that the N-terminal region of the polypeptide corresponds to a predicted discoidin-like domain. This domain architecture is conserved in fungal muskelins and reveals a structural parallel between the muskelins and certain extracellular fungal galactose oxidases, although the phylogeny of the two groups appears distinct. In view of the fact that a number of kelch-repeat proteins have been shown to self-associate, co-immunoprecipitation, protein pull-down assays and studies of cellular localization were carried out with wild-type, deletion mutant and point mutant muskelins to investigate the roles of the discoidin-like and kelch-repeat domains. We obtained evidence for cis- and trans-interactions between the two domains. These studies provide evidence that muskelin self-associates through a head-to-tail mechanism involving the discoidin-like domain.