Eucalyptus adult material requires more suc-cessive subcultures in the in vitro multiplication phase for increased vigor and cellular activity. This study evaluated the endophytic manifestation and shoot multiplication of one 13-year-old Eucalyptus benthamii clone under differ-ent culture conditions and used canopy branches (CB) and trunk base material as explant sources. The culture media were wood plant medium (WPM), Murashige and Skoog medium (MS) and JADS (Correia and co-authors medium). Based on the results of the initial multiplication experi-ment, further tests examined sucrose concentrations and pH. Morphophysiology, dry mass production, endophytic manifestation and histochemical were determined. Explant sources responded differently to MS and JADS media, but the WPM medium promoted homogeneous development. The responses were similar for both explant sources when sucrose concentrations varied. Shoots died in the absence of sucrose, showed high oxidation at 60 g L-1 and optimal development at 30 g L-1. Endophytes were more evident for shoots from the CB origin. Explant sources responded distinctively to treatment due to physiological and intrinsic genetic factors. Therefore, explant sources, different cul-ture media, sucrose concentration and pH may determine micropropagation success and influence the presence and/or intensity of endophytic manifestation.