Intraspecific variation in the composition of three cytotoxic secondary metabolites from the New Zealand marine sponge Mycale hentscheli collected at two sites in central New Zealand was quantified by 1H NMR techniques. A total of 275 sponges were analyzed bimonthly over 15 mo to compare intersite (approximately 100 km) and intrasite (approximately 100 m) spatial and temporal variations in the metabolites. Biological and physical characteristics of sponge size, morphology, depth, and temperature were recorded at each site. Metabolite concentrations were found to vary in space and time. Metabolite composition was site-specific; mycalamide A, pateamine, and peloruside A were present at Pelorus Sound, whereas pateamine was absent from sponges at Kapiti Island. Pateamine and peloruside A concentrations in sponges at Pelorus Sound varied seasonally; no such patterns were observed at Kapiti Island. Relationships of compound concentration with volume and depth were complex. High levels of peloruside A in Pelorus Sound sponges from between 8 and 10 m depth coincided with a density boundary layer and chlorophyll a maximum.