Mice lacking the glutamate receptor subunit delta2 (GluRdelta2) are deficient in cerebellar long-term depression (LTD) at the parallel fibre-Purkinje cell synapses. We conducted delay and trace eyeblink conditioning with these mice, using various temporal intervals between the conditioned stimulus (CS) and unconditioned stimulus (US). During trace conditioning in which a stimulus-free trace interval (TI) of 250, 100 or 50 ms intervened between the 352-ms tone CS and 100-ms US, GluRdelta2-mutant mice learned as successfully as wild-type mice. Even in the paradigm with TI = 0 ms, in which the end of CS and onset of US are simultaneous, there was no difference between the GluRdelta2-mutant and wild-type mice in their acquisition of a conditioned response. However, in the delay paradigm in which the 452-ms CS overlapped temporally with the coterminating 100-ms US, GluRdelta2-mutant mice exhibited severe learning impairment. The present study together with our previous work [Kishimoto, Y., Kawahara, S., Suzuki, M., Mori, H., Mishina, M. & Kirino, Y. (2001) Eur. J. Neurosci., 13, 1249-1254], indicates that cerebellar LTD-independent learning is possible in paradigms without temporal overlap between the CS and US. On the other hand, GluRdelta2 and cerebellar LTD are essential for learning when there is CS-US temporal overlap, suggesting that the cerebellar neural substrates underlying eyeblink conditioning may change, depending on the temporal overlap of the CS and US.