This study was performed in order to determine how monocytes and macrophages in co-culture with autologous head and neck squamous cell carcinoma (HNSCC) tumor tissue regulate the secretion of monocyte chemotactic protein-1 (MCP-1). The levels of MCP-1 were measured when autologous monocytes or monocyte-derived macrophages (MDMs) were co-cultured in vitro with autologous fragment (F)-spheroids established from HNSCC tumors or benign mucosa serving as control. MCP-1 secretion from co-culture stimulated monocytes and MDMs was increased compared to spontaneous MCP-1 secretion. With prolonged co-culture, MDMs showed a steady-state MCP-1 secretion above background levels for up to 96 h, even with change of co-culture media every 24 h. Addition of an anti-MCP-1 antibody to the medium decreased co-culture-induced monocyte IL-6 secretion. Addition of lipopolysaccharide (LPS) (1 [microg/ml) reduced MCP-1 secretion compared to spontaneous secretion in monocyte cultures. F-spheroids also secrete MCP-1, but at insignificant levels compared to the MCP-1 secretion from monocytes and MDMs. MCP-1 secretion from monocytes/MDMs is regulated differently when co-culture stimulation is compared to LPS-stimulation. Monocytes and MDMs expressed MCP-1 mRNA at a high level in all tested conditions: stimulated in co-culture, not stimulated or stimulated with LPS, indicating post-transcriptional regulation of MCP-1 secretion. The secretion of MCP-1 from tumor-derived F-spheroids, and the maintenance of co-culture MCP-1 secretion from MDMs in vitro, suggests that tumor-associated macrophages are a source of MCP-1 in HNSCC tumors.