The mammalian thalamus is composed of two types of thalamocortical relay. First order relays receive information from subcortical sources and relay it to cortex, whereas higher order relays receive information from layer 5 of one cortical area and relay it to another. Recent reports suggest that modulatory inputs to first and higher order relays may differ. We used rat thalamic brain slices and whole cell recordings from relay cells in various first order (the lateral geniculate nucleus, the ventral posterior nucleus, and the ventral portion of the medial geniculate body) and higher order (the lateral posterior, the posterior medial nucleus, and the dorsal portion of the medial geniculate body) relays to explore their responses to activation of muscarinic receptors. We found that, whereas all first order relay cells show a depolarizing response to muscarinic activation, approximately 20% of higher order relay cells respond with hyperpolarization. The depolarization is accompanied by an overall increase in input resistance, whereas the hyperpolarization correlates with a decrease in resistance. Because activation of cholinergic brain stem afferents to thalamus increases with increasing behavioral vigilance, the findings suggest that increased vigilance will depolarize all first order and most higher order relay cells but will hyperpolarize a significant subset of higher order relay cells. Such hyperpolarization is expected to bias these relay cells to the burst firing mode, and so these results are consistent with evidence of more bursting among higher order than first order relay cells.