C ultured peripheral blood leukocytes (PBL) from individuals homozygous or heterozygous for the defective gene causing the inherited disease cystic fibrosis (CF) secrete three different ciliary dyskinesia substances (CDS), which can be detected by their activity in vitro in a rabbit mucociliary bioassay. Their PBL also release substances that promote mucus expulsion and destruction of the ciliated epithelium. In the present study the relative numbers of lymphocytes (T, B, and null), monocytes-macrophages (Mφ), and polymorphonuclear neutrophils were found to be normal in subjects with the CF gene, as were the responses of their PBL to phytohemagglutinin and pokeweed mitogen. Using purified subpopulations of leukocytes, we obtained evidence that both monocytes and T lymphocytes can secrete CDS in vitro with no requirement for cooperation with other lymphocyte subsets, whereas B and “null” lymphocytes probably require either differentiation or cellular cooperation for optimal secretion of CDS. Mucus expulsion and tissue destruction were produced by substances released primarily from polymorphonuclear neutrophils and secondarily from Mφ. Using cycloheximide and actinomycin D, we obtained evidence that CDS accumulation requires active protein synthesis and is not dependent on newly synthesized RNA, at least in short-term cultures. Gel filtration chromatography of active culture supernates showed that T lymphocytes synthesized only a CF-specific CDS, whereas Mφ synthesized all three CDS found in PBL cultures. Evidence is presented that one CDS is related structurally to C3a, since it can be removed with rabbit antisera specific for human C3a.