ABSTRACT The objective of this study was to evaluate the effect of auxin pulse intervals on the induction of somatic embryos of Eucalyptus grandis x E. urophylla and to describe the embryogenic behavior of callus under the effect of auxinic stress. Cotyledons were inoculated in culture medium containing 207.07 µM picloram, a treatment considered as auxin pulse. Explants that were in the auxin pulse treatment were transferred to semisolid or liquid medium containing 20.71 µM picloram after one, two, four or eight days of auxin pulse. In a second experiment, explants that were on auxin pulse treatment were transferred to semi-solid medium containing 20.71 µM picloram after one, two or three days of auxin pulse. Auxiliary picloram pulse treatments (207.02 µM) can be used as an initial source of stress for the acquisition of embryogenic competence. The oxidation of cotyledonary explants may be considered as an indication of the formation of embryogenic calli. The presence of pectins in peripheral regions of somatic pro-embryos can be considered as a marker of somatic embryogenesis in cotyledonary explants of Eucalyptus grandis x E. urophylla.