In A7r5 cells, vasopressin (AVP) and 5-hydroxytryptamine (5-HT) share the ability to stimulate phospholipase C and so production of inositol 1,4,5-trisphosphate (IP3). AVP can also reciprocally regulate capacitative and non-capacitative Ca²⁺ entry pathways via its ability to stimulate nitric oxide synthase-III (NOS-III), but 5-HT appears not to activate NOS-III and stimulates only capacitative Ca²⁺ entry (Moneer et al. 2005). The IP3-evoked Ca²⁺ release promoted by AVP causes a long-lasting inhibition of adenylyl cyclase (AC) dependent on activation of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) (Dyer et al. 2005). These observations suggest rather precise associations between receptors, Ca²⁺ signalling pathways, and proteins that are regulated by Ca²⁺. Our aim was to examine the associations between these signalling proteins. cDNA was synthesised from RNA and quantitative real-time PCR (RT-PCR) was performed. Each reaction included LUX primers for a specific CaMKII or AC subunit and primers for a housekeeping gene (beta-actin). Non-detergent discontinuous sucrose gradients were used to separate membrane fractions (Ostrom et al. 2002). Immunoprecipitation of solubilized membranes was performed using appropriate primary antibodies and Protein A/G Plus-agarose beads. Proteins in immunoprecipitates or sucrose gradient fractions were identified by immunoblotting. Proteins were separated by SDS-PAGE on 4-12% Bis-Tris gradient gels, transferred to PVDF membranes, blocked and incubated with primary and secondary antibodies using standard methods. All experiments were performed at least 3 times. Immunoblotting established that AC3, AC5/6, CaMKII, type 1 and type 3 IP3 receptors and NOS-III were expressed in A7r5 cells. From RT-PCR, d-CaMKII was the major expressed isoform, and AC3 and AC6 were each expressed at similar levels. Both V1A receptors for AVP and 5-HT2A receptors were expressed predominantly in non-caveolar membranes, whereas NOS-III and type 1 and 3 IP3 receptors were more abundant in fractions that included caveolae. Immunoprecipitation established an association between caveolin 3 and IP3 receptors, caveolin 3 and NOS-III, and between type 3 IP3 receptors and CaMKII. Our results suggest that the selective ability of AVP, but not 5-HT, to activate NOS-III is unlikely to result from colocalization of V1A receptors with NOS-III in caveolae. An association of type 3 IP3 receptors with CaMKII may underlie the selective ability of IP3-evoked Ca²⁺ signals to inhibit AC activity via CaMKII.