The presence of positive acute phase proteins within the circulation of rheumatoid arthritis patients suggests that elevated cytokine production associated with this chronic inflammatory disorder initiates the hepatic acute phase response. Cytokines produced at inflammatory lesions are believed to travel via the circulation to the liver where they induce acute phase protein production by hepatocytes. To test whether serum from rheumatoid arthritis patients contained sufficient levels of cytokines to promote an acute phase response in vitro, a bioassay was developed that employed the human hepatoma cell line Hep3B. These cells produced the acute phase protein serum amyloid A (SAA) in response to a combination of recombinant IL-1 beta and IL-6 or to monocyte conditioned medium. Serum (or plasma) from normal individuals or from rheumatoid arthritis patients did not induce SAA production by Hep3B cells. Moreover, these serum samples did not prevent SAA production induced by monocyte conditioned medium, indicating that they did not contain inhibitors of cytokine activity. Despite the inactivity of serum samples, synovial fluid samples obtained from rheumatoid arthritis patients were active in the hepatocyte bioassay and promoted SAA synthesis. One synovial fluid sample was analysed in detail to identify cytokines responsible for the SAA-inducing activity. Neutralizing antisera against IL-6 and IL-1 beta blocked this activity by > 90% whereas anti-IL1 alpha and anti-TNF-alpha sera were without effect. Absolute cytokine levels within the synovial fluid sample were determined by ELISA; IL-6, IL-beta and TNF-alpha, but not IL-1 alpha, were confirmed to be present. Moreover, the synovial fluid sample contained a large amount of the IL-1 receptor antagonist. These data indicate, therefore, that synovial fluid recovered from an inflamed joint contains all the necessary cytokines in balance with inhibitors to promote SAA production by Hep3B cells. The steady state levels of these factors within the plasma compartment, however, were insufficient to induce the acute phase response by cultured Hep3B cells, suggesting that this system does not mimic the relationship between the circulation and the liver that likely exists in rheumatoid arthritis patients.