Female sexual attraction to male urine is elicited by darcin. Total time spent under test male stimulus (blue bars: male urine; cyan bars: recombinant major urinary protein (MUP) alone; hatched bars: male urine plus recombinant MUP) and matched control stimulus (pink bars: BALB/c female urine; open bar: buffer), together with the difference in time spent under test minus control (circles), plotted as means ± standard error of mean. Significant P values indicate greater attraction to the test male stimulus [matched pair t-tests of log transformed data (t)]. Control tests using intact urine from C57BL/6 strain [B6, A(a)] or a random selection of n = 14 wild males [Wild, B(f)] confirmed greater attraction to male urine. No attraction was shown towards BALB/c male urine [Bc, B(g)] which contained an extremely low level of darcin. Recombinant darcin (r-darcin, 11 μg) stimulated significant attraction when presented alone [A(b,c)] and when added to male BALB/c urine [B(h)]. There was no attraction to other recombinant MUPs (r-18694Da; r-18645Da). SDS-PAGE of urine stimuli (C), equivalent to one-thirtieth of the amount used in behavioural tests. The different mobility of r-darcin relative to native darcin is a consequence of the C-terminal His-tag used for purification (see Additional File 1: Figure S4).