Prostate cancer cells metastasise to bone causing a predominantly osteosclerotic response. It has previously been shown that PC3 cells secrete factors which stimulate the mitogenic activity of human bone marrow stromal (hBMS) cells. Some of these mitogens have been found to be proteins with a molecular weight between 20 and 30 kDa. Even though a number of investigations have been performed to identify the osteoblastic mitogenic factor or factors produced by prostate cancer cells, it is still unknown what causes the mitogenic activation of osteoblasts. Therefore, the aim of this study was to characterise the protein profile of conditioned medium (CM) from PC3 cells in the molecular weight range of 5-30 kDa using proteome analysis. A protein profile of the CM from PC3 cells was performed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Thirty protein spots with molecular weights ranging from 5 to 30 kDa were analysed by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). One of these spots was identified as cyclophilin A. We examined whether cyclophilin A alone or in combination with insulin-like growth factor-I (IGF-I) had any effects on the proliferation or differentiation of hBMS cells. Cyclophilin A at 1, 10, and 100 nM and cyclophilin A at 10 nM combined with 10 ng/ml IGF decreased the proliferation of hBMS cells up to 49+/-30, 38+/-29, 50+/-8 and 60+/-16%, respectively [mean (treated/control)+/-standard error of the means (SEM)] of control. IGF-I did not significantly affect these decreases. Cyclophilin A alone or in combination with IGF-I did not have any effect on differentiation (assessed by measuring the activity of alkaline phosphatase (ALP)). In conclusion, these results suggest cyclophilin A is not involved in the osteosclerotic effects seen when prostate cancer metastasises to bone.