ABSTRACT: This study investigated changes in the electroencephalograph (EEG) power spectrum as well as physiological and behavioral responses to on-farm killing via mechanical cervical dislocation (MCD), manual cervical dislocation (CD) or intravenous pentobarbital sodium administration in lightly anesthetized laying hens, to evaluate the welfare impact of each method. A mixed group of 44 white Leghorn and Smoky Joe laying hens (60 weeks-old) were anesthetized with isoflurane in oxygen and maintained at 1.5-2% isoflurane/O2 until the killing method was applied. Birds were randomly assigned to one of three experimental groups on each trial day. The EEG was recorded bilaterally in a four-electrode montage. After recording a 5-min baseline, the killing method was applied and EEGs and other behavioral and physiological responses, including convulsions, gasping, cessation of body movements and feather erection were recorded for 5 min. Changes in EEG frequency bands (alpha, beta, delta, theta), median frequency (F50), 95% spectral edge frequency (F95), and total power (Ptot) were used to assess the quality of the on-farm killing event. Within 15 s after administration of pentobarbital sodium, there were significant decreases in mean frequency bands, increases in mean F50 and F95, and decreases in Ptot, suggesting brain death. In addition, birds presented a shorter latency to cessation of movement after pentobarbital sodium injection compared to MCD and CD (22 vs. 115 s and 136 s, respectively). There were significant increases in F95 and decreases in Ptot at 120 s after application of CD; and a concomitant decrease in the frequency bands at 135 s and isoelectric EEG at 171 ± 15 s. Changes consistent with brain death after MCD included isoelectric EEG at 207 ± 23 s and a significant decreases in some frequency bands at 300 s post-application. No other significant spectrum frequency changes were observed in the MCD group, suggesting brain death likely occurred near the 5-min endpoint. There was no clear association between behavioral, physiological, and EEG responses within CD and MCD treatments. The data demonstrate that pentobarbital sodium induced a rapid death with minimal behavioral and physiological responses regardless of strain of hens. In comparison, use of CD and MCD resulted in a slow onset of brain death in hens.