The synthesis and release of feline leukemia virus p30 was studied using a permanently infected feline thymus tumor cell line. Disrupted cells were divided into two subcellular fractions, a cytoplasmic extract (CE) representing cellular material soluble in 0.5% NP-40 and a particulate fraction (PF) insoluble in 0.5% NP-40 but soluble in 0.2% deoxycholate and 0.5% NP-40. Intracellular feline leukemia virus p30 was isolated from infected cells by immune precipitation with antiserum to p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the precipitated proteins. Cells labeled for 3 h with [35S]methionine contained equal amounts of p30 in both the CE and the PF. p30 synthesis was estimated to be 0.8% of the total host cell protein synthesis. Immune precipitates from cell pulse labeled for 2.5 min contained a labeled 60,000-dalton polypeptide (Pp60) in the PF and a polypeptide in the CE that comigrated with feline leukemia virus p30 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When cells were chased after a pulse label, there was a rapid loss of Pp60 in the PF and an accumulation of p30 in the CE within 30 min followed by distribution of p30 in both the PF and the CE. Estimation of intracellular and extracellular p30 levels during a 0.5- to 24-h chase period suggested that most of the newly synthesized p30 was incorporated into extracellular virus. Typtic peptide analysis of labeled Pp60 and p30 demonstrated the presence of 13 of 15 p30 peptides within the Pp60 molecule. The tryptic peptide analysis in concert with the pulse-chase labeling data provides strong evidence that Pp60 is a precursor of p30.