Granule-poor human neutrophil cytoplasts, prepared without heat or cytochalasin B treatment so as to preserve both motile function and activatable respiratory burst oxidase, were investigated for their content of several isoforms of protein kinase C (PKC). Immunoblotting with isoform-specific rabbit antibodies (Abs) to PKC revealed that both the alpha-specific and the beta(I and II)- specific Abs recognized a protein band of 78 kd comigrating with PKC from rat brain cytosol. The gamma-specific antiserum did not detect any protein of this molecular mass. The cytoplast beta-PKC band was more readily detected than the cytoplast alpha-PKC band. Antibodies to beta I- or beta-II- specific PKC sequences showed the beta II subtype to be the predominant form of beta-PKC, although some beta I was also found. The identity of the 78-kd cytoplast bands as PKC was established by the fact that phorbol ester treatment of intact cytoplasts induced translocation of the bands from cytosol to membrane fractions. However, whereas PKC specific activity was similar in cytoplast lysates and brain cytosol, immunoreactivity of cytoplast alpha- and beta-PKC bands was considerably less than that of rat brain. Hydroxylapatite chromatography of partially purified cytoplast PKC revealed two major peaks of PKC activity precisely coeluting with brain alpha- and beta-PKC and displaying comparable enzymatic activities despite the relatively weak immunoreactivity of cytoplast alpha- and beta-PKC. To our knowledge, this is the first demonstration that human neutrophil-derived cytoplasts contain alpha, beta I, and beta II forms of PKC and that each isoform translocates from cytosol to membrane upon exposure to phorbol ester at concentrations that induce superoxide production. In addition, our evidence raises the possibility that cytoplasts may also possess other isoforms of PKC that we are unable to detect with our alpha, beta, and gamma antibodies. Finally, the granule-poor cytoplasts seem a particularly useful preparation in which to examine the role of individual PKC isoforms in neutrophil activation.