1. Achatin-I (Gly-d-Phe-l-Ala-l-Asp), a neuroactive tetrapeptide having a d-phenylalanine residue, has been proposed to be an excitatory neurotransmitter of Achatina giant neurons. It was revealed that the d-Phe 2 residue is essential for bioactivity of achatin-I, which seems to adopt β-turn conformation. In the present study, in order to investigate the structure-activity relationships of achatin-I and its derivatives, the two highly constrained analogs of achatin-I, [Δ 2 Phe 2 ]achatin-I (Gly-Δ 2 Phe-l-Ala-l-Asp) (Δ 2 Phe: (Z)-α,β-dehydrophenylalanine) and [Aib 2 ]achatin-I (Gly-Aib-l-Ala-l-Asp) (Aib: α-aminoisobutyric acid), were synthesized, and their effects on the two identifiable Achatina giant neuron types, PON (periodically oscillating neuron) and v-RCDN (ventral-right cerebral distinct neuron), were examined in comparison with those of achatin-I under voltage clamp.2. Achatin-I (n = 6), ejected onto the neurone by brief pneumatic pressure (2 kg/cm 2 , 400 ms, 10 - 3 M, at 10-min intervals), produced an inward current (I i n ) on PON. The I i n value (mean ± SEM) was 0.44±0.03 nA. The interval between the achatin-I ejection and the I i n peak was 14.74 ± 3.15 s (n = 6). [Δ 2 Phe 2 ]achatin-I (n = 6) and [Aib 2 ]achatin-I (n = 6) had no effect on this neuron type.3. On the other hand, achatin-I (n = 10) and [Δ 2 Phe 2 ]-achatin-I (n = 10), ejected by brief pressure, produced an I i n on v-RCDN. The I i n values were 0.85±0.07 nA for achatin-I and 0.48±0.05 nA (p<0.01, compared with that of achatin-I by Student's t-test for paired data) for [Δ 2 Phe 2 ]achatin-I. The intervals between the compound ejection and the I i n peak were 5.95±0.33 s for achatin-I and 8.70±0.81 s (p<0.05, compared with that of achatin-I) for [Δ 2 Phe 2 ]achatin-I. [Aib 2 ]achatin-I (n = 10) had no effect on this neuron type.