Abstract. Two of the three major outer layers of the Chlamydomonas reinhardtii cell wall (W6 and W4) can be solubilized from living cells with sodium perchlorate or other chaotropes and will repolymerize in vitro to form milligram amounts of wall crystals. Conditions for optimal crystalization are presented, and conditions that fail to induce polymerization are exploited to maintain monomers in aqueous solution for ionexchange chromatography. The four major glycoproteins of the complex (GP1, 1,5, 2, and 3) have in this way been purified to apparent homogeneity and have been characterized morphologically by transmission electron microscopy using the quick-freeze, deepetch technique and by amino acid composition. Three of the four are hydroxyproline-rich species that copolymerize to form the W6 layer. The fourth (GP1.5) is a glycine-rich species that binds to the interior of the in vitro crystal; it is apparently equivalent to the granules within the W4 layer in situ.T I~E extracellular matrices of both plant and animal cells are, by definition, assembled extracellularly, meaning that all the information necessary for the ultimate form of a given matrix is contained within its component proteins and polysaccharides. In some cases, the best studied being type I collagen (2), the matrix protein is secreted as a precursor that can only assemble after being processed by extracellular enzymes; in other cases (9,14), the patterning of the matrix appears to be accomplished, at least in part, by the sequential secretion of different components. Much experimental evidence (34) indicates that cellular differentiation is often accompanied, or predicted, by alterations in the extracellular matrix, and that the dedifferentiation events that accompany malignant transformation can include matrix alterations (5). Therefore, an understanding of matrix assembly has widespread implications.We have chosen to study the assembly of the extracellular matrix of Chlamydomonas reinhardtii, a unicellular eukaryote, because it offers unique advantages as an experimental system. (a) The matrix is highly organized and invariant, and several of its layers are fundamentally two-dimensional (31), permitting analysis of its organization that becomes far more difficult with three-dimensional arrays. (b) Two of the layers can be disassembled using chaotropic agents and will reassemble in vitro when the chaotropes are removed (13, 17), permitting critical analysis of the assembly process by methods used, for example, in studies of collagen (2). (c) The glycoproteins that make up the matrix are fibrous hydroxyproline-rich species that are likely to be evolutionary homologues of the major matrix proteins of higher plants (21,30), and are at least analogues of the hydroxyproline-containing collagens of higher animals; therefore, information about their assembly promises to increase our understanding of multicellular interactions. In a previous paper (13), we described the overall architecture of the Chlamydomonas cell wall as visualized using the quick-...