The primary structure of the zeta subspecies of rat brain protein kinase C was deduced from its overlapping cDNAs. The zeta subspecies of protein kinase C consists of 592 amino acid residues with the calculated molecular mass of 67,740 Da and has regulatory and protein kinase domains in its amino- and carboxyl-terminal halves, respectively. Although all members of the protein kinase C family so far identified have a tandem repeat of the characteristic cysteine-rich zinc-finger-like sequence in the regulatory domain, the zeta subspecies contains only one set of this sequence. Northern (RNA)-blot hybridization analysis indicated that two major RNA transcripts of the zeta subspecies with different lengths may be generated by the use of different polyadenylylational signals. The enzyme was expressed in COS-7 cells by transfection with the cDNA construct encoding its whole sequence. It showed an approximate molecular mass of 64,000 Da upon SDS/PAGE. The enzyme activity was significantly dependent on phospholipid but was independent of the presence of Ca2+ or diacylglycerol, when assayed with calf thymus H1 histone as a phosphate acceptor protein. The zeta subspecies expressed in COS-7 cells did not appear to show binding activity of phorbol ester. The structural and biochemical properties indicate that the zeta subspecies is related to, but distinct from, other subspecies of protein kinase C. Perhaps, this subspecies belongs to another entity of the enzyme family.