Microinjection of the excitatory amino acid glutamate is commonly used to stimulate neuronal cell bodies in brainstem nuclei that are crucial for cardiovascular regulation, respiratory control, and other functions. One such nucleus, the rostral ventrolateral medulla (RVLM), integrates afferent information to coordinate cardiovascular responses to changes in the environment. In the RVLM, an increase in mean blood pressure of ≥25 mmHg following glutamate microinjection is widely accepted as evidence of accurate localisation of this nucleus. However, the dose of glutamate, and injection volume, varies considerably between investigators, and the optimal parameters are controversial. Here we examined data from 34 publications over the past 20 years in which glutamate doses, ranging from 0.0051 to 8.5 μg, were injected to identify the rostral ventrolateral medulla (RVLM) in rat. The aim of this chapter is to describe, in a pragmatic way, the ideal parameters for this method. Our meta-analysis reveals that there is a dose–response relationship between glutamate and blood pressure at low doses, but once the dose is sufficient to elicit a �35 mmHg rise in blood pressure, the response plateaus (∼0.5 μg). Interestingly, the injection volumes used in the studies examined do not show any correlation with the observed blood pressure responses. Neither strain nor weight of rat had any influence on the blood pressure response induced by glutamate. Glutamate microinjection is a stable and reproducible method for activating cell bodies, but not fibres of passage, in most brain regions, and may also be a useful tool for normalising other drug responses.