The habituation of the acoustic startle reflex (ASR) was examined concerning individual differences in sensitivity to punishment (PUN) and sensitivity to reward (REW), within the general framework of the reinforcement sensitivity theory (RST) of personality. Two hypotheses derived from the RST were evaluated: the separable subsystems hypothesis and the joint subsystems hypothesis. In addition, we examined the direction of the relationship of PUN and REW with the habituation of the ASR. A habituation segment of electromyography recordings of the orbicularis oculi was assessed with an unconditional latent curve model. In accordance with the RST hypotheses, the relationship of PUN and REW on the habituation process was assessed with two conditional latent curve models. There was higher support for the separable subsystems hypothesis. In addition, PUN and REW related with the habituation trajectory of the ASR in the expected directions. Higher levels of PUN and lower levels of REW related with a slower habituation of the ASR, whereas lower levels of PUN and higher levels of REW related with a faster habituation of the ASR.