A low molecular weight (approximately 16,000), early protein is characterized as the product of the essential T4 head assembly gene 31. This gene is known to be required to allow formation of any ordered head structure from the major T4 capsid protein, P23 (Laemmli, U.K., Beguin, F., and Gujer-Kellenberger, G. (1970) J. Mol. Biol. 47, 69-85). In wild type infection P31 synthesis ceases at late times; in contrast, P31 is overproduced in certain early or regulatory T4 mutant infections, particularly gene 55 mutant infections. P31 was purified preparatively from Escherichia coli infected with the latter mutant, but could only be obtained for the most part in modified form, possibly due to unusual sensitivity to a proteolytic activity. P31 is not cleaved in vivo during normal head assembly, nor does it become a part of the mature head or any ordered prehead structure as determined by an immunological assay using antiserum prepared against the purified protein. However P31 does appear to become a part of the unordered P23 aggregates (lumps) which accumulate when ordered P23 assembly is prevented. We cound find no evidence for P31 association with T4 DNA or the host membrane. Our experiments favor the hypothesis that P31 directly affects the aggregation state and solubility properties of P23.