To search for new urinary protein biomarkers of cadmium toxicity, we used quantitative two-dimensional electrophoresis (2DE) and analysed urine samples from 18 male cadmium recovery plant employees whose mean age was 47 +/- 15.6 years (+/- 1 SD) and whose urine cadmium levels ranged from 0.14 microgram l-1 to 20.4 micrograms l-1 (0.06-37.1 micrograms g-1 creatinine). Image analysis of the silver-stained gels yielded intensity (concentration) values for a mean number, per person, of 825 +/- 184 urinary proteins (spots) and found 596 +/- 218 matched proteins (the same proteins in two or more gels) per person. Total urinary protein and the sum of all spot intensities were positively correlated (P = 0.0447 and P = 0.0616, respectively) with urinary cadmium (UCD), as measured by atomic absorption spectroscopy. The combined sum of the intensities of all acidic proteins with a relative molecular weight (M(r)) below 40 kDa was correlated with UCD (P = 0.0461), revealing a low M(r), acidic proteinuria as UCD increased. Multiple hypothesis testing by regression analysis of the intensities of matched proteins with UCD revealed 14 unidentified proteins that were considered candidates for biomarkers of cadmium exposure. The best two candidate proteins--those having M(r)s of less than 13.9 kDa and relative glyceraldehyde-3-phosphate dehydrogenase (G3PDHr) coordinates of -19.7 and -27.2--were excellently resolved in the 2DE gels, and their intensities increased by 323% and 857%, respectively, over the UCD range that was tested. Two other proteins with M(r)s of 23.9 kDa and 29.2 kDa and with acidic net charges were not as well resolved. Six very acidic proteins, with M(r)s ranging from 88.8 to 90.7 kDa and with intensities highly correlated with UCD, appeared to be related and were resolved as a 'charge train' (a group of related proteins, or isoforms, differing only by small changes in net charge). Four proteins appeared to increase only when the UCD concentration was above a threshold of 16 micrograms l-1.