The high molecular weight basic nuclear proteins from winter flounder sperm are a group of at least 16 proteins which are on average 1000 amino acids long. Despite their large size and complexity, this group of proteins gave rise to only 20 major peptides when digested with trypsin, suggesting that they are closely related to each other and contain sequences which are repeated internally many times over. When the phosphorylated forms of the high molecular weight basic nuclear proteins were isolated from mid-spermatid nuclei and digested with trypsin, six of the major peptides were shown to be phosphorylated. Five of these phosphopeptides were purified, and sequenced by automated Edman degradation. Each of the five contained all or part of the consensus sequence X-Ser(P)-X-Ser(P)-Pro, where X represents lysine or arginine. These phosphorylation sites which may be repeated an average of 20 times in each of the high molecular weight proteins, are different from the phosphorylation sites seen in other basic nuclear proteins. The dephosphorylated high molecular weight basic nuclear proteins from flounder sperm are phosphorylated in vitro at these sites by mid-spermatid nuclei.