MALDI-TOF imaging mass spectrometry (IMS) is used to examine the deposition of trisoxazole macrolides in the tissues of the Penares nux sponge. IMS showed that kabiramides, the major trisoxazole macrolides in P. nux, were allocated preferentially in the external tissues of the sponge. The deposition covered the outer tissue layers from the pinadoderm through mesohyl and ectosome to the outer portion of the choanosome. The presence of each macrolide varied independently from one sponge specimen to others, and showed no inductive or suppressive influences on each other compounds nor particular preferences on any specific cell types or tissues. The results provided a new and additional evidence into the specificity of the sponge's toxic metabolite distribution in the tissue level. The outward distribution of the toxic macrolides in P. nux aligned with the optimal defense theory, and suggested that the sponge has evolved its chemical defensive mechanisms purposely for the best protection of the most vulnerable, newly generated branches of the colony.