Intranasal instillation techniques are used to deliver various substances to the upper and lower respiratory tract (URT and LRT) in mice. Here, we quantify the relative distribution achieved with intranasal delivery of a nonabsorbable tracer, 99m Tc-labeled sulfidecolloid. Relative distribution was determined by killing mice after instillation and quantifying the radioactivity in dissected tissues using gamma scintigraphy. A significant effect of delivery volume on relative distribution was observed when animals were killed 5 min after instillation delivered under gas anesthesia. With a delivery volume of 5 l, no radiation was detected in the LRT; this increased to a maximum of 55.7 Ϯ 2.5% distribution to the LRT when 50 l were delivered. The majority of radiation not detected in the LRT was found in the URT. Over the course of the following 1 h, radiation in the LRT remained constant, while that in the URT decreased and appeared in the gastrointestinal tract. Instillation of 25 l into anesthetized mice resulted in 30.1 Ϯ 6.9% distribution to the LRT, while only 5.3 Ϯ 1.5% (P Ͻ 0.05) of the same volume was detected in the LRT of awake mice. Varying the body position of mice did not affect relative distribution. When using intranasal instillation, the relative distribution between the URT and LRT and the gastrointestinal tract is heavily influenced by delivery volume and level of anesthesia. gamma scintigraphy; topical treatment; upper respiratory tract; lower respiratory tract THE ADMINISTRATION OF SUBSTANCES to mice by the intranasal route is an effective, noninvasive technique employed for the delivery of allergens (3, 4, 26), drugs or gene therapy (4, 20), immunotherapy (1, 7, 11), and pathogens (12,16,18) to the upper and lower respiratory tracts (URT and LRT). In published studies, volumes of substances intranasally instilled into mice range from 5 l (22) to 100 l (3), with little justification for the chosen volumes. Intranasal delivery is often carried out after intraperitoneal (9, 19) or inhalation (5, 14, 15) anesthesia but has also been performed with fully awake mice (1,6,17). The position of the mouse during intranasal delivery has also varied between studies, with horizontal (13) and head-down supine (12) positions having been used. The specific delivery protocols used in these studies are thought to influence the relative distribution of the delivered substance to the URT, LRT, and gut. However, to our knowledge, very little published information is available describing the distribution of intranasally delivered substances or how the distribution can be influenced by delivery techniques.Preliminary studies by Tsuyuki et al. (23) have shown that 75% of a 50-l dose of intranasally administered Evans blue dye is deposited in the airways, with no dye detectable in the esophagus or stomach. Eyles et al. (7) reported that 48% of a 50-l dose of intranasally instilled 7-m-diameter 46 Sc-labeled styrene-divinyl benzene microspheres was evident in the lungs 15 min after challenge, while Takafuji et al. (21) ...