Methane (CH4) generated by cattle is both a major source of greenhouse gas emissions and a powerful indicator of feed conversion efficiency; thus, accurate quantification of CH4 production is required for addressing future global food security without neglecting environmental impacts. A newly developed Ruminant Emission Measurement System (REMS) supports research on the relationships between bovine nutrition, genetics, and management strategies by measuring eructated CH4 emissions from ruminal activity. REMS is a substantial improvement and extension of the chamber technique, which is considered the standard method to quantify ruminant CH4 generation. Part I of this two-part series describes the design and evaluation of REMS. An uncertainty analysis of chamber emission rate (ER) was conducted to identify critical measurement component contributions to overall ER uncertainty and guide component selection. In Part II, REMS commissioning was performed and a method for system validation including overall emission uncertainty is reported. REMS consists of six positive pressure ventilated hood-type chambers individually equipped with a thermal environmental control subsystem, fresh air supply control subsystem, and gas sampling subsystem. Estimates of the standard uncertainty for each measurement parameter were quantified and propagated through the ER equation derived from CH4 and air mass flow balances. A sensitivity analysis was conducted to assess the contribution of each parameter to the emission rate standard uncertainty (absolute = ΔER; relative = %ΔER) under predicted normal operation by varying gas analyzer and ventilation measurement uncertainties as anticipated with REMS use. Results showed that expanded %ΔER (~95% confidence level) associated with the methane ER computation was approximately 5.9% for ER values between 3.5 and 17.2 g h-1. Ventilation rate and gas concentration measurements were the major sources of uncertainty, contributing about 69% and 29%, respectively, to the uncertainty associated with methane ER values. This work provides the foundation for future studies using respiration chambers to include a stated standard uncertainty associated with animal emission measurements.