Utilizing laser scanning confocal, fluorescence resonance energy transfer (FRET), and atomic force (AFM) microscopy, we have investigated the association of PKCa with microtubules during stimulus-induced relocalization in A7r5 smooth muscle cells. Confocal microscopy using standard immunostaining techniques confirmed earlier observations that colchicine disruption of microtubules blocked PKC localization in the peri-nuclear region of the cell caused by phorbol 12, 13 dibutyrate (PDBu, 10^-6M). Dual-immunostaining suggested co-localization of PKC and -tubulin in both unstimulated and PDBu-treated cells. This finding was verified by FRET microscopy which indicated that the association of PKC was heterogeneous in distribution and confined primarily to microtubules in the peri-nuclear region. FRET analysis further showed that the association between the molecules was not lost during colchicine-induced dissolution of microtubules, suggesting the formation of tubulin/PKC complexes in the cytosol. Confocal imaging indicated that peri-nuclear microtubular structure was more highly sensitive to colchicine dissolution compared to other regions of the cell. Topographical imaging of fixed cells by AFM indicated a well-defined elevated structure surrounding the nucleus which was absent in colchicine-treated cells. It was calculated that the volume of the nuclear sleeve-like structure of microtubules increased approximately 5-fold in PDBu-treated cells, suggesting a probable increase in microtubular mass. Based on PKC localization, increased colchicine sensitivity, and their volume change in stimulated cells, the results suggest that peri-nuclear microtubules form a specialized structure which may be more dynamically robust than in other regions of the cell. PKC could contribute to this dynamic activity. Alternatively, peri-nuclear microtubules could act as a scaffold for regulatory molecule interaction at the cell center.