In bovine ciliary muscle (BCM) cells, we have previously shown that stimulation of M₃-muscarinic receptor (MR₃) opens two types of receptor-operated non-selective cation channel (ROC) which serve as major pathways for Ca²⁺ entry during the tonic phase of contraction (Takai et al. 2004). We have also shown by RT-PCR that BCM (whole tissue) contains mRNA for four TRP channel homologues (TRPC 1, 3, 4 and 6), commonly regarded as molecular candidates for ROCs (see Inoue et al. 2003, for review). In the present experiments we tried to visually identify these TRPCs in the plasma membrane of BCM by immunofluorescence microscopy. Smooth muscle cells were isolated by treatment with collagenase and papain from the ciliary body dissected out from bovine eyes obtained from a local slaughterhouse. A CCD fluorescence microimaging system (Olympus Co., Tokyo, Japan) was used. After 4-5 days culture of 150-300 BCM cells on the centre of a fibronectin-coated round glass plate (14 mm diameter) in serum-free HAM F12 media (Sigma), the body of the cells was removed by gentle sonication under hypotonic conditions. The plasma membrane remaining attached on the glass surface was treated with polyclonal primary antibodies (2.5-5 μg/ml; Chemicon Co., Temecula, CA, USA; catalogue codes: AB5446, AB5576, AB5812 and AB5914) against putative cytoplasmic segments of the four TRPCs and visualized with secondary antibodies labelled with an Alexa Fluor 546 fluorescent dye (Invitrogen; peak absorption and emission at 556 and 573 nm, respectively). The membrane preparations were also similarly immunostained with antibodies against MR₃ (Biogenesis Ltd, Poole, UK; cat. number: 6391-2150) and α-actin (Progen Biotechnik GmbH, Heidelberg, Germany; cat. number, 65001). All the anti-TRPC antibodies used gave many microscopically visible spots of immunofluorescence (roughly 1 spot/μm²) in the regions of the cell-free membrane preparation which were also positively stained with antibodies against MR₃ and α-actin. Essentially similar results were obtained in at least three replicated experiments for each of the four anti-TCPC antibodies. The existence of the four TRPC homologues in the plasma membrane of the BCM was thus clearly demonstrated. Much fewer fluorescent spots were detected in the membrane regions devoid of α-actin staining, indicating a relatively sparse distribution of the TRPC homologues in non-smooth muscle cells (such as fibroblasts) in the ciliary muscle tissue. The present results encourage further study to examine the possible relationship between TRPCs and muscarinic receptor-operated cation channels.