The contribution of mossy fiber sprouting to the generation of spontaneous seizures in the epileptic brain is under dispute. The present study addressed this question by examining whether sprouting of mossy fibers is present at the time of appearance of the first spontaneous seizures in rats, and whether all animals with increased sprouting have spontaneous seizures. Epileptogenesis was induced in 16 rats by electrically stimulating the lateral nucleus of the amygdala for 20-30 min until the rats developed self-sustained status epilepticus (SSSE). During and after SSSE, rats were monitored in long-term by continuous video-electroencephalography until they developed a second spontaneous seizure (8-54 days). Thereafter, monitoring was continued for 11 days to follow seizure frequency. The density of mossy fiber sprouting was analyzed from Timm-stained preparations. The density of hilar neurons was assessed from thionin-stained sections. Of 16 rats, 14 developed epilepsy. In epileptic rats, the density of mossy fiber sprouting did not correlate with the severity or duration (115-620 min) of SSSE, delay from SSSE to occurrence of first (8-51 days) or second (8-54 days) spontaneous seizure, or time from SSSE to perfusion (20-63 days). In the temporal end of the hippocampus, the sprouting correlated with the severity of neuronal damage (ipsilateral: r = -0.852, P < 0.01 contralateral: r = -0.748, P < 0.01). The two animals without spontaneous seizures also had sprouting. Increased density of sprouting in animals without seizures, and its association with the severity of neuronal loss was confirmed in another series of 30 stimulated rats that were followed-up with video-EEG monitoring for 60 d. Our data indicate that although mossy fiber sprouting is present in all animals with spontaneous seizures, its presence is not necessarily associated with the occurrence of spontaneous seizures.