It is possible to cause G 2 arrest in Aspergillus nidulansby inactivating either p34 cdc2 or NIMA. We therefore investigated the negative control of these two mitosis‐promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34 cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34 cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non‐dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34 cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature‐sensitive mutations cause an arrest in G 2 at 42 C. Addition of MMS to nimT23 G 2‐arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 C and this delay was correlated with a long delay in the dephosphorylation and activation of p34 cdc2. Addition of MMS to nimA5 G 2‐arrested cells caused inactivation of the H1 kinase activity of p34 cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 C. However, if Tyr15 phosphorylation of p34 cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G 2‐arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 C. Thus, Tyr15 phosphorylation of p34 cdc2 in G 2 arrests initiation of mitosis after DNA damage in A. nidulans.