Protein phi 0 is a unique protein which is present in the sperm of the sea cucumber, Holothuria tubulosa. It associates with histones, but its physiological role is unknown. From its amino acid composition and sequence, protein phi 0 can be considered as an H1-related protein. In this paper, we have studied its interaction with chicken erythrocyte chromatin particles of different complexity, from core particles to polynucleosomes. Addition of protein phi 0 results in marked chromatin insolubilization. The higher the molecular weight of the chromatin fragment, the lower is the phi 0/nucleosome molar ratio at which precipitation occurs, so that complete insolubilization of polynucleosomes is achieved at a phi 0/nucleosome molar ratio which is identical to that found in mature H. tubulosa spermatozoa. We have also found that the interaction of protein phi 0 with chromatin is cooperative. These findings contribute to clarification of the peculiar physico-chemical properties shown by H. tubulosa sperm chromatin and the role played by the phi 0 protein.