We describe the characterization of a spontaneously transformed chicken monocytic cell line that developed as a single colony of cells in a heterophil culture that was inadvertently left in the incubator over a period of 25 days. These cells, hitherto named HTC, grow efficiently at both 37 or 41 degrees C in culture medium containing either 5% FBS or 2% chicken serum. The HTC cells are acid phosphatase positive, show expressions of both class I and class II major histocompatibility complex (MHC), CD44, K1, and K55 cell surface antigens, and engulf latex beads, produce nitrite and interleukin-6 on stimulation with bacterial lipopolysaccharide (LPS). Treatment with phorbol myristate acetate (PMA) induces respiratory burst in HTC cells and the secretion of matrix metalloproteinase (MMP) into culture medium. Using gene-specific primers and reverse transcriptase-polymerase chain reaction (RT-PCR), the presence of mRNA trancripts for interferon-gamma (IFN-gamma), interleukin-1 (IL-1), interleukin-6 (IL-6), nitric oxide synthase (NOS), matrix metalloproteinase-2 (MMP-2), and transforming growth factor-beta (TGF-beta) were detected. Lipopolysaccharide (LPS) treatment of HTC cells modulated IL-1, IL-6, IFN-gamma, NOS mRNA levels as detected by RT-PCR analyses. Using different avian tumor virus gene-specific primers and PCR, the HTC cells were positive for the presence of avian leukosis virus (ALV) and Marek's disease virus (MDV) but negative for reticuloendothelial virus (REV), chicken infectious anemia virus (CIAV), and herpes virus of turkeys (HVT). The production of ALV antigens by HTC cells was further confirmed using p27 gag protein ELISA. Collectively, these results show that the HTC cells belong to myeloid/macrophage lineage and were likely transformed by ALV and MDV but retain many interesting and useful biological activities.