This report is part of a project to characterize cotton gin emissions from the standpoint of stack sampling. In 2006, EPA finalized and published a more stringent standard for particulate matter with nominal diameter less than or equal to 2.5 µm (PM2.5). This created an urgent need to collect additional cotton gin emissions data to address current regulatory issues, because current EPA AP-42 cotton gin PM2.5 emission factors did not exist. The objective of this study was the development of PM2.5 emission factors for cotton gin 2nd stage seed-cotton cleaning systems based on the EPA-approved stack sampling methodology, Method 201A. The project plan included sampling seven cotton gins across the cotton belt. Key factors for selecting specific cotton gins included: 1) facility location (geographically diverse), 2) industry representative production capacity, 3) typical processing systems, and 4) equipped with properly designed and maintained 1D3D cyclones. Five of the seven gins were equipped with 2nd stage seed-cotton cleaning systems. In terms of capacity, the five gins were typical of the industry, averaging 33.8 bales/h during testing. Some test runs were excluded from the test averages because they failed to meet EPA Method 201A test criteria. Also, other test runs included in the analyses had cotton lint fibers that collected in the ≤ 10 µm and/or ≤ 2.5 µm samples. This larger lint material can impact the reported emissions data, but EPA Method 201A does not suggest methods to account for these anomalies. Average measured 2nd stage seed-cotton cleaning system PM2.5 emission factor based on the five tests (12 total test runs) was 0.0037 kg/227-kg bale (0.0080 lb/500-lb bale). The 2nd stage seed-cotton cleaning system average emission factors for PM10 and total particulate were 0.038 kg/bale (0.084 lb/bale) and 0.063 kg/bale (0.138 lb/bale), respectively. The 2nd stage seed-cotton cleaning system PM2.5 emission rate from test averages ranged from 0.089 to 0.16 kg/h (0.20-0.36 lb/h). System average PM10 and total particulate emission factors were lower than those currently published in EPA AP-42. The ratios of 2nd stage seed-cotton cleaning system PM2.5 to total particulate, PM2.5 to PM10, and PM10 to total particulate were 5.8, 9.5, and 61.1%, respectively.