Neurotoxin microinjected into the retrotrapezoid nucleus of anesthetized rats decreases phrenic activity and eliminates the response to CO₂. In unanesthetized rats, such treatment has no effect on awake, resting breathing and decreases CO₂ sensitivity by 40% (M. Akilesh, M. Kamper, A. Li, and E. E. Nattie. J. Appl. Physiol. 82: 469-479, 1997). One important factor in explaining these disparate results is the actual size of the anatomic lesion. In the present study, we injected ibotenic acid into the retrotrapezoid nucleus of anesthetized rats and evaluated lesion size by using two new approaches: 1) DEAD red, a fluorescent probe that enters impaired cells through leaky membranes and binds to nucleic acids, and 2) conjugation of toxin to fluorescent beads. With the use of DEAD red, the region containing labeled dying cells was 313 ± 104 nl (n = 4), six times larger than the initial injected volume, and the physiological effects on phrenic amplitude, the CO₂ response, and blood pressure began within minutes and were substantial. With conjugated toxin, in theory, neuronal damage would be limited to the region of detectable fluorescence (49 ± 10 nl; n = 4). Effects on phrenic amplitude, CO₂ sensitivity, and blood pressure were absent until ~2 h postinjection. Control experiments, with 2 h of in vitro incubation of the neurotoxin-microbead conjugate and injection of the supernatant after centrifugation, showed similar results that suggest release of conjugated neurotoxin. We conclude that DEAD red provides a useful means to monitor neuronal impairment in acute studies in vivo. Conjugation of neurotoxin to microbeads may be less reliable in this regard.