Hybridoma cell lines secreting monoclonal antibodies which bind beef heart cytochrome c or Paracoccus denitrificans cytochrome c have been produced using spleen cells from BALB/c mice immunized with cytochrome c. Immunization was performed with either the native cytochrome c, succinylated hemocyanin-conjugated cytochrome c, or beef heart cytochrome c polymerized with glutaraldehyde. Of 10 such fusions, the hybridization frequency ranged from 0 to 42%. The cell fusion efficiency, the possible factors involved in the cell fusion efficiency and the frequency of antibody producing hybridomas are described. The percentage of hybridomas positive for anti-cytochrome c antibody production as screened for by radioimmunoassay or ELISA was 2%. Of the antibodies from 12 hybridoma cell lines which resulted from 10 fusions, three were specific to beef heart cytochrome c, another three were specific to P. denitrificans cytochrome c, and the remainder reacted with both cytochromes c. These groups of monoclonal antibodies react to different sets of sites on these two cytochromes c. The monoclonal antibodies from ten representative clones have been isolated and characterized by different methods.