BACKGROUND: Although acupuncture sensation (also known as de qi) is a cornerstone of traditional acupuncture therapy, most research has accepted the traditional method of defining acupuncture sensation only through subjective patient reports rather than on any quantifiable physiological basis. PURPOSE: To preliminarily investigate the frequency of key sensations experienced while needling to specific, quantifiable tissue levels (TLs) guided by ultrasound (US) imaging. METHODS: Five participants received needling at two acupuncture points and two control points at four TLs. US scans were used to determine when each TL was reached. Each volunteer completed 32 sets of modified Southampton Needle Sensation Questionnaires. Part one of the study tested sensations experienced at each TL and part two compared the effect of oscillation alone versus oscillation+rotation. RESULTS: In all volunteers, the frequency of pricking, sharp sensations was significantly greater in shallower TLs than deeper (p=0.007); the frequency of sensations described as deep, dull and heavy, as spreading, and as electric shocks was significantly greater in deeper TLs than shallower (p=0.002). Sensations experienced did not significantly differ between real and control points within each of three TLs (p>0.05) except TL 4 (p=0.006). The introduction of needle rotation significantly increased deep, dull, heavy sensations, but not pricking and sharp sensations; within each level, the spectrum of sensation experienced during both oscillation+rotation and oscillation alone did not significantly differ between acupuncture and control points. CONCLUSION: The preliminary study indicates a strong connection between acupuncture sensation and both tissue depth and needle rotation. Furthermore, the new methodology has been proven feasible. A further study with an objective measurement is warranted.