HDS and LDS rats are the result of selective breeding for differences in the hypothermic effects of the 5‐hydroxytryptamine‐1A (5‐HT1A) agonist 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT); HDS (high DPAT sensitivity) rats exhibit a much greater hypothermic response than do LDS (low DPAT sensitivity) rats. It is possible that this genetically‐based difference in sensitivity to the hypothermic effects of the 5‐HT1Aagonist is associated with a change in other behaviours modulated by 5‐HT neurotransmission. The present study examined the acoustic startle response, the classically conditioned enhancement of startle, and the effects of 8‐OH‐DPAT and buspirone treatments on these measures, in HDS and LDS rats. On four test sessions, HDS and LDS rats were exposed to 20 acoustic startle stimuli (115 dB; 40 ms in duration). For each test session, 10 trials were presented in the dark (Noise Alone trials) and 10 were presented at the end of a 3500 ms presentation of a 15 W signal light (Light + Noise trials). LDS rats exhibited greater startle amplitude than did HDS rats on Noise Alone trials. Initially, there was no difference in startle amplitude on the Light + Noise versus Noise Alone trials in either LDS or HDS rats. By the end of the first test session, however, and continuing throughout the remainder of the four test sessions, startle amplitude on the Light + Noise trials was significantly greater than in the Noise Alone trials. The magnitude of this startle‐potentiated startle (SPS) effect did not differ in HDS versus LDS rats. SPS testing was continued for three additional sessions; in these sessions the effects of acute treatment with the 8‐OH‐DPAT (125 μg/kg, subcutaneously (s.c.)), the novel anxiolytic buspirone (4 mg/kg, intraperitoneally (i.p.)) or vehicle (distilled water) were determined. Both 8‐OH‐DPAT and buspirone treatment increased baseline (Noise Alone) startle amplitude in LDS rats but not in HDS rats. With respect to the conditioned enhancement of startle, buspirone reduced the SPS effect in both HDS and LDS rats, whereas 8‐OH‐DPAT did not change the conditioned enhancement effect in either rat line. These findings suggest that the selective breeding for differences in 8‐OH‐DPAT‐induced hypothermia has resulted in changes in other behaviours and also changes in the response to 5‐HT1Aagonist treatment. Moreover, these findings are consistent with the hypotheses that: (a) 5‐HT1Aagonist actions underlie the buspirone‐induced and 8‐OH‐DPAT‐induced increases in Noise Alone startle amplitude; whereas (b) the buspirone‐induced reduction in potentiated startle is not the result of 5‐HT1Aagonist actions of this compound.