Uteri of Lif null mice do not support embryo implantation. Since deletion of some genes often prevents the survival of null mice to adulthood, we have used a proven inhibitor of leukaemia inhibitory factor (LIF) signalling to identify the precise window of time during which LIF is required in vivo, and assessed the cellular expression of several LIF-associated targets. On day 4 of pregnancy, mice were injected with hLIF-05 (inhibitor) into the uterine lumen, with corresponding volumes of PBS (vehicle) injected into the contralateral horn. On days 5 and 6, the number of implantation sites was recorded and the uteri processed for immunohistochemistry. Blockade of LIF on day 4 reduced embryo implantation by 50% (P≤0.0001) and was effective maximally between 0930 and 1230 h. Antagonism of LIF signalling was evidenced by a lack of phosphorylated STAT3 in the luminal epithelium (LE). Amphiregulin was absent from the LE on day 4 evening and H-type-1 antigen expression was retained in the LE on day 5 in inhibited uteri. Interleukin-1α and oncostatin M expression were reduced in the stroma on day 6, following LIF inhibition. Unexpectedly, PTGS2 expression in stroma was unaffected by LIF inhibition in vivo, in contrast to Lif null mice. In summary, this suggests that LIF signalling is effective for implantation during a discrete time window on day 4 and antagonism of LIF signalling recapitulates many features exhibited in Lif null uteri. The data presented validates the use of antagonists to investigate tissue specific and temporal cytokine signalling in reproductive function. © 2009 Society for Reproduction and Fertility.