Trimethylamine oxide (TMAO) is a common osmolyte and counteracting solute. It is believed to combat the denaturation induced by hydrostatic pressure as some deep-sea animals contain higher TMAO levels than their shallow water counterparts. It has also been proposed that TMAO may accumulate passively during lipid storage resulting in a correlation between lipid content and TMAO levels in some groups. Previous research showed that lipid content decreased with depth in species of Hawaiian fishes presenting a novel test of these competing hypotheses. TMAO ranged from 20.4 to 92.8 mmol/kg. Lipid content ranged from 0.50 to 4.7% WW. After completing a comprehensive search for depths available in the literature, provided here, we analyzed TMAO and lipid as a function of average, minimum and maximum depth of occurrence for 27 species of fishes from nine orders. We found that TMAO is positively correlated with all measures of habitat depth (hydrostatic pressure) but the relationship is strongest with average depth. We further showed using phylogenetic independent contrasts that this relationship was not influenced by the evolutionary relatedness of these species. Interestingly, we found that lipid content increased with depth, in direct contrast to previous studies. TMAO is thus also positively correlated with lipid content. While we are unable to distinguish between these hypotheses, we show that TMAO is strongly correlated with depth in mid-water fishes.