Sea urchin spermatozoa contain a unique histone H1 (SpH1) with an unusual primary structure. Within 12 minutes postinsemination SpH1 is phosphorylated and lost from the chromatin. Both sperm and egg kinases phosphorylate SpH1 in vivo. Until now the analysis of the phosphorylation of SpH1 in eggs in vivo during the first 12 minutes has been impossible because of the enormous volume of the egg relative to the sperm nucleus. Here we present the first such analysis based on the direct extraction of [32P]-labeled SpH1 from eggs, fractionation by HPLC, cleavage by CNBr and isolation of the two CNBr-generated fragments of SpH1 by HPLC. Two dimensional maps of the [32-P]-peptides show that at least five sites in the amino-terminal fragment, and five sites in the carboxyl-terminal fragment, are phosphorylated by 1 minute postinsemination.