The effects of the temporal sequence of tone (conditioned stimulus [CS]) and footshock (unconditioned stimulus [US]) during training (acquisition) on tone-dependent retention were studied in mice. Freezing increased significantly as an associative behavioral response in mice subjected to CS paired with US or after unpaired by 30 s in the memory test performed 24 hr after training. In the home cage of freely moving mice implanted with an electrocardiogram transmitter, CS triggered a strong tachycardiac response in the memory test. Heart rate (HR) increased from about 580 bpm to more than 750 bpm, and HR variability decreased significantly. The inhibition of the HR increase by the nonspecific beta-adrenergic antagonist sotalol indicated the strong sympathetic contribution to the tachycardiac response. CS evoked a significant but minor HR increase in mice subjected to either CS or US only or CS and US unpaired by 60 s. Thus, HR and HR variability reflected associative learning.