We have greatly improved the yield after cell fusion of antigen-specific monoclonal antibody-secreting murine hybridomas by substitution of Sp2/0 ascites for fetal bovine serum (FBS) in the culture medium. As a medium supplement for established cultures, Sp2/0 ascites at 2.5% in Dulbecco's modified Eagle's medium (DMEM) provided growth characteristics similar to media containing 10% or 20% FBS. All culture parameters associated with hybridoma fusion experiments, however, were advantageously affected in ascites-supplemented cultures. Clonal growth of hybridomas from the single cell stage was enhanced at least two-fold over 20% FBS-supplemented medium. Following fusion, both the number of colonies and hybridoma growth rates were substantially increased for ascites-containing cultures. Most importantly, the number of antigen-specific antibody-secreting hybridomas was increased in Sp2/0 ascites supplemented cultures, five-fold in the eight fusion experiments presented here. This improved performance compared to FBS-supplemented medium is reproducible from lot to lot of ascites.