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 1st 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. All seven gins had 1st stage seed cotton cleaning systems. In terms of capacity, the seven gins were typical of the industry, averaging 30.0 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 1st stage seed-cotton cleaning system PM2.5 emission factor based on the seven tests (19 total test runs) was 0.0083 kg/227-kg bale (0.018 lb/500-lb bale). The 1st stage seed-cotton cleaning system average emission factors for PM10 and total particulate were 0.074 kg/bale (0.162 lb/bale) and 0.101 kg/bale (0.222 lb/bale), respectively. The 1st stage seed-cotton cleaning system PM2.5 emission rate from test averages ranged from 0.15 to 0.37 kg/h (0.32-0.82 lb/h). System average PM10 emission factors were higher and system average total particulate emission factors were lower than those currently published in EPA AP-42. The ratios of 1st stage seed-cotton cleaning system PM2.5 to total particulate, PM2.5 to PM10, and PM10 to total particulate were 8.3, 11.3, and 73.3%, respectively.