Nocturnal activity patterns of northern myotis (Myotis septentrionalis) at diurnal roost trees remain largely uninvestigated. For example, the influence of reproductive status, weather, and roost tree and surrounding habitat characteristics on timing of emergence, intra-night activity, and entrance at their roost trees is poorly known. We examined nocturnal activity patterns of northern myotis maternity colonies during pregnancy and lactation at diurnal roost trees situated in areas that were and were not subjected to recent prescribed fires at the Fernow Experimental Forest, West Virginia from 2007 to 2009. According to exit counts and acoustic data, northern myotis colony sizes were similar between reproductive periods and roost tree settings. However, intra-night activity patterns differed slightly between reproductive periods and roost trees in burned and non-burned areas. Weather variables poorly explained variation in activity patterns during pregnancy, but precipitation and temperature were negatively associated with activity patterns during lactation.