The effects of urea structural analogues on the urea-facilitated diffusion system were examined in human red cell membranes (pink ghosts) and in antidiuretic hormone(ADH)-stimulated frog urinary bladder epithelia. In both tissues, urea permeability (P(urea)) was dramatically but reversibly inhibited by a number of urea analogues, such as 1-(3,4-dichlorophenyl)-2-thiourea (DCPTU). This urea derivative reduced the urea flux in a dose-dependent manner (90% inhibition of P(urea) at 0.5 mM concentration of DCPTU). With the aim of obtaining irreversible markers of red cell and urinary bladder urea transport systems, urea derivatives were modified by addition of an azido residue (N3) and preliminary experiments of photoaffinity labelling were carried out. Two synthetic urea derivatives: 1-(3-azido-4-chlorophenyl)-2-thiourea (ACPTU) and 1-(3-azido-4-chlorophenyl)-3-methyl-2-thiourea (Me-ACPTU) were shown to be very potent inhibitors of P(urea) when used in the absence of light, with IC50 values 60.3 microM and 31.6 microM respectively, as measured in frog urinary bladder. Both these molecules appeared to bind covalently to the urea carrier in both frog urinary bladder and human pink red cell ghosts, when illuminated in the presence of the tissue: the urea flux, which fell to 30-70% of the value obtained in the presence of ADH after inhibitor addition, remained low after the preparation had been illuminated for 30 min and the inhibitor removed. These results provide an interesting approach to the urea carrier analysis, particularly to the urea or urea analogue binding site on the transport protein.