Fluorescent derivatives of the bisindolylmaleimide inhibitors of protein kinase C (PKC) were synthesized and tested with respect to their inhibitory potency, specificity, and usefulness as fluorescent cytological stains for PKC. Several of the fluorescent bisindolylmaleimide derivatives (fim-1, fim-2, and rim-1) acted as ATP-competitive catalytic site inhibitors and retained much of the potency and specificity of the parental compound. The R6-C1 and the PKC beta 1-overexpressing R6-PKC3 cell lines were used for testing fim-1 and rim-1 as cytological stains for PKC. Comparisons showed that the R6-PKC3 cells stained much more brightly than R6-C1 cells. When R6-PKC3 cells were treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) for 30 min, staining with fim-1 or anti-PKC beta 1 revealed a dramatic translocation of PKC to the cell periphery. When R6-PKC3 cells were exposed to PMA for 24 h to down-regulate PKC, cytoplasmic staining was drastically reduced. Staining patterns obtained with an antibody specific for PKC beta 1 and with fim-1 were remarkably similar except for mitochondrial staining, which was only seen with fim-1. A closer examination of the mitochondrial staining showed that mitochondria convert from filamentous to punctate shapes and cluster around the nucleus when cells are treated with PMA. This punctate morphology, perinuclear clustering, and staining with fim-1 persists when PKC is down-regulated. Overall, these results indicate that fim-1 and rim-1 can serve as useful fluorescent probes for PKC. The mitochondrial staining may be due to a PKC isoform resistant to down-regulation.