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 α-specific and the β(I and II)-specific Abs recognized a protein band of 78 kd comigrating with PKC from rat brain cytosol. The γ-specific antiserum did not detect any protein of this molecular mass. The cytoplast β-PKC band was more readily detected than the cytoplast α-PKC band. Antibodies to βI- or β-II-specific PKC sequences showed the βII subtype to be the predominant form of β-PKC, although some βI 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 α- and β-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 a- and β-PKC and displaying comparable enzymatic activities despite the relatively weak immunoreactivity of cytoplast α- and β-PKC. To our knowledge, this is the first demonstration that human neutrophil–derived cytoplasts contain α, βI, and βII 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 α, β, and γ antibodies. Finally, the granule-poor cytoplasts seem a particularly useful preparation in which to examine the role of individual PKC isoforms in neutrophil activation.