Factors affecting hybridoma yield following in vitro immunization have been investigated. Of critical importance for optimum yield was the batch of young calf serum used both in immunization cultures and in post-fusion cultures. Only 1 batch of serum was found to be suitable for the immunization step. The addition of horse serum to deficient young calf serum in pre-fusion cultures did not reconstitute the essential component(s). Addition of T cell, macrophage and bovine endothelial cell conditioned medium to the supportive batch of young calf serum in pre-fusion cultures did not increase the yield of hybridomas. For one antigen (insulin) the yield of hybrid cells was dependent on the concentration of antigen in immunization cultures. This was not the case, however, with the second antigen (ACTH) within the concentration range examined. The optimum spleen cells density was 1 X 10(7) cells/ml and the optimum culture period before fusion was 5-6 days. Although in vivo priming followed by in vitro boosting decreased the yield of hybrids the relative percentage of positive hybrids was slightly increased. The results suggest that in vivo priming is not essential since primary in vitro stimulation alone produced significant numbers of hybrids secreting specific antibody.