Blood monocytes from healthy volunteers were isolated by Ficoll-Isopaque centrifugation and cultured (together with lymphocytes) in medium 199 with 20% heat-inactivated newborn calf serum in a Teflon culture bag. Quantifiable data on survival showed that up to 21 days of culture, approximately 40% of the initial number of monocytes were still viable. Such cultures could be maintained for more than 8 weeks without refeeding. The monocytes exhibited the morphology of macrophages after 5-7 days of culture, and increased in size during culture. Less than 1% of the cells became giant cells even after long culture periods. Almost all cultured monocytes were positive for alpha-naphthyl butyrate esterase, whereas the peroxidase-positive granules disappeared during the first week of culture. After long culture times increasing amounts of lysozyme and angiotensin-converting enzyme were detected in the culture supernatants. Phagocytosis of staphylococci did not decrease appreciably during culture, and the same holds for intracellular killing of these bacteria. Chemotactic activity decreased during culture, whereas the chemokinetic response of the monocytes persisted.