Conflicting data have been published on whether low-dose methotrexate (MTX) treatment of rheumatoid arthritis (RA) is able to slow down radiological joint damage, i.e. retard the destruction of articular cartilage and (subchondral) bone. We studied the effects of MTX on proteoglycan (PG) turnover and interleukin-1 (IL-1)- and RA mononuclear cell (RA-MNC)-induced cartilage damage in human articular cartilage tissue cultures, and the effects of MTX on basal and RA-MNC-influenced proliferation and differentiation of osteoblasts in cultures of human bone-derived osteoblasts. MTX exerted no direct effect on cartilage nor did MTX influence IL-1- or RA-MNC-induced cartilage damage, despite strong suppression of basal as well as mitogen- and antigen-induced RA-MNC proliferation. MTX induced strong inhibition of osteoblast proliferation, but did not significantly interfere with osteoblast differentiation (i.e. alkaline phosphatase activity). RA-MNC-enhanced proliferation and differentiation of osteoblasts were abolished by MTX. These results suggest that if MTX is able to induce retardation of radiological progression in RA, this is not based on an initial direct effect of MTX on cartilage as measured by PG turnover, nor on an initial inhibition of IL-1- or RA-MNC-induced cartilage damage. However, longstanding MTX-induced inhibition of RA-MNC proliferation may lead to reduction of the catabolic activity involved in cartilage destruction. On the other hand, long-term inhibition of osteoblast proliferation may eventually lead to decreased bone formation and osteopenia. Whether this will turn out to be a problem of clinical importance in the treatment of RA has to be established.