In vitro DMSP-lyase activity (DLA) measurements were made during an Atlantic Meridional Transect (AMT) cruise in May 2004, which travelled through the southern and northern oligotrophic gyres. Along the transect, twice-daily measurements were taken from the surface (0.5 − 8.0 m) and the chlorophyll maximum (chl max; 47 − 150 m). Surface samples showed a large variation in DLA (0 − 58.6 nmol L−1 h−1), while values for the chl max were lower and less variable (0 − 8.7 nmol L−1 h−1). A negative correlation was observed between DLA:TChl a and primary production (carbon fixation rate) in surface samples (Spearman’s ρ = −0.57, p < 0.05, n = 17). Nitracline depth and the ratio of photo-protective to photosynthetic accessory pigments indicate that, as expected, surface phytoplankton communities in the gyres experience low nutrient concentrations and high light levels in comparison to communities from the chl max. Comparing pre-dawn and late-morning surface DLA values provides a pseudo time-series for each day and, in areas with elevated DLA, values for the late-morning samples were consistently higher than those taken at pre-dawn. Our field data set covers a geographically extensive area and includes the first DLA analyses from the oligotrophic South Atlantic gyre.