The Leishmania mexicana CRK3 gene encodes a cdc2-related protein kinase with activity towards histone H1. Attempts to disrupt both alleles of CRK3 in the promastigote life-cycle stage resulted in changes in cell ploidy, which were avoided only when an extra copy of CRK3 was expressed from an episome. This provides strong evidence that CRK3 is essential to L. mexicana. The cyclin-dependent kinase specific inhibitor flavopiridol inhibited affinity purified histidine tagged CRK3 (CRK3his) with an IC(50) value of 100 nM and inhibited in vitro growth of L. mexicana promastigotes. Incubation of promastigotes with 2.5 microM flavopiridol for 24 h led to cell cycle arrest with an accumulation of 95% of cells in G2 or early mitosis (G2/M). Release from cell cycle arrest resulted in a semi-synchronous re-entry into the cell cycle; samples taken at 2, 4, and 6 h after release from the block were enriched for cells in G1 (68%), S-phase (70%), and G2/M phase (61%), respectively. This method of synchronisation was used to show that the majority of CRK3his activity towards the substrate histone H1 was present at G2/M. These data suggest that CRK3 has an essential role in controlling cell cycle progression at the G2/M-phase transition in L. mexicana promastigotes.