We sought to determine the presence of Fos-like immunoreactive (Fos-LI) cells in the pontine brainstem following cholinergically induced sustained rapid-eye movement (REMc) sleep in cats. Microinjections (0.25 microliter) of vehicle (N = 2) or carbachol (2.0 micrograms/0.25 microliter; N = 4) were made into the medial pontine reticular formation. Carbachol produced a state with all the signs of natural REM sleep and with durations of 15.2-57.8 min. Compared with vehicle control animals, carbachol treated animals showed a significantly higher number of Fos-LI cells in pontine regions implicated in REM sleep generation, with longer REMc bouts associated with more Fos-LI cells than the short-duration bout. Regions with REMc-associated Fos-LI increases included: the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei, where some Fos-LI cells were immunohistochemically identified as cholinergic; the locus coeruleus, where some of the Fos-LI cells were identified to be catecholaminergic; the dorsal raphe and the pontine reticular formation. These findings suggest immediate early gene activation is associated with the ubiquitous biological state of REM sleep.