Calcium plays an important regulatory role in olfactory signal transduction. Many investigations into the regulation of the olfactory signaling pathway have been performed using fractions enriched in ciliary membranes from olfactory sensory neurons. The traditional method of preparing ciliary fractions uses high calcium concentrations, thought to dislodge cilia from the dendritic knobs of the olfactory neurons in the nasal epithelium. However, calcium, an important second messenger in the odorant signaling cascade, modulates the activity of many enzymatic reactions in this cascade. Pre-exposure of cilia to high calcium concentrations may modify these signaling events. Therefore, we sought to develop a method of isolating cilia-enriched membranes that avoids exposing the cilia to high calcium concentrations. Our method of isolation, referred to as the mechanical agitation method, involves mechanical disruption and sonication of the olfactory epithelium to dislodge the cilia. To evaluate this method of cilia preparation, basal adenylyl cyclase activity, as well as forskolin- and odorant-activated adenylyl cyclase, were analyzed. Specific activity of adenylyl cyclase and protein yield were compared for the mechanical agitation and the high calcium preparations. Immunoblots were analyzed for the presence of transduction components enriched in olfactory cilia: adenylyl cyclase type III (ACIII), heterotrimeric G-protein subunit Galphaolf and the 1 C2 isoform of phosphodiesterase (PDE 1 C2). Based on these analyses, the ciliary fraction prepared by the mechanical agitation method appears to be very similar to that prepared by the high calcium method, with a higher yield.