In fission yeast the Nim1 kinase phosphorylates and inactivates the cdc2-inhibitory Weel tyrosine kinase. In addition, Nim1 is necessary for an efficient cellular response to nutritional starvation leading to cell cycle arrest in G1. Given the remarkable evolutionary conservation of the cell cycle regulatory mechanism we have investigated the effect of Nim1 expression on the control of the mammalian cell cycle using a plasmid microinjection approach. In synchronised IMR90 human fibroblasts, expression of Nim1 strongly inhibited entry into S-phase. This effect was dependent on the catalytic activity of Nim1 and did not require its regulatory domain. Furthermore we show that co-expression of human Wee1 kinase reverted the inhibitory effect, indicating that Nim1 was acting in a Wee1-dependent manner. These results provide evidence for the existence of a Nim1-like kinase pathway acting at the G0-1/S transition in human cells.