Using hamster anti-Pichinde virus serum, we immunoprecipitated polypeptides from BHK-21 cells infected with Pichinde virus. Seven immunoprecipitable polypeptides exhibited a time- and multiplicity of infection-dependent appearance when the cultures were pulse-labeled with L-[35S]methionine for 1 h. The predominant polypeptide was a nucleoprotein (NP) of 64,000 daltons. Components of 48,000, 38,000, and 28,000 daltons, when analyzed by two-dimensional tryptic peptide mapping, were found to be derived from NP. After a 3-h chase period, polypeptides of 17,000, 16,500, and 14,000 daltons were evident, and peptide mapping revealed that these three polypeptides were also related to NP. During a series of pulse-chase experiments, a 79,000-dalton glycoprotein (GPC) was cleaved to glycoproteins of 52,000 and 36,000 daltons. Radiolabel in a polypeptide of approximately 200,000 daltons (L) did not chase into smaller cleavage products. L, GPC, and NP were found to be unique by two-dimensional tryptic peptide mapping. Comparison of polypeptides immunoprecipitated from infected cells with structural components of purified virus revealed that L protein was evident in both. This is the first report of a high-molecular-weight polypeptide in Pichinde virus particles and infected cells.