Using single unit nerve recording techniques in rats, the present experiment aimed to determine which specific population of afferent nerve fibers (groups I, II, III and IV) in the dorsal roots at the 4th or 5th lumbar segments (L4 or L5) are activated during manual acupuncture needle stimulation. An acupuncture needle 300–340 µm in diameter was inserted into the skin and underling muscles around the Zusanli acupoint (ST36) area in the hindlimbs, and was manually rotated right and left at a frequency of about 1 Hz for 1 min. The dorsal root of the L4 and L5 spinal nerve was cut close to the entrance into the spinal cord after laminectomy and dissected free to record unitary afferent nerve activity. A single afferent fiber activated by acupuncture stimulation was identified by the identical shape of the discharge spikes during stimulation and during electrically evoked action potentials induced by single pulse electrical stimulation of the sciatic nerve. The conduction velocity of the afferent fiber was calculated by the latency of the electrically evoked action potential. A total of 35 units were intentionally recorded from all animals in order to include all 4 afferent fiber groups. Units were spontaneously silent in the absence of stimulation, while all units responded to ipsilateral manual rotation of the acupuncture needle. The conduction velocity of all 35 units ranged between 0.8 and 86.0 m/s, thus belonging to groups I–V fibers. Mean conduction velocity of groups I, II, III and IV were 57.9 m/s (n = 13), 42.9 m/s (n = 11), 10.3 m/s (n = 6) and 1.2 m/s (n = 5), respectively. Mean discharge rates during acupuncture stimulation of groups I, II, III and IV afferents were 7.4 Hz, 6.2 Hz, 4.7 Hz and 0.4 Hz, respectively. Discharge rates of group IV afferent fibers were significantly lower than those of groups I, II and III afferents. It was concluded that manual acupuncture needle stimulation to the hindlimbs activated afferent nerve fibers belonging to all four groups of afferents in rats. It is suggested that all four groups of somatic afferents activated by manual acupuncture stimulation will elicit various effects when action potentials are delivered to the central nervous system.