Cultured isolates of Prochlorococcus from the Mediterranean Sea (MED4) and Sargasso Sea (SS120) have been shown to have dramatically different pigment composition and growth rate responses when grown over a range of irradiances. Moreover, analyses of field populations in the North Atlantic have shown that distinct ecotypes can coexist in the same water column. These and other observations have led to the hypothesis that Prochlorococcus is comprised of genetically distinct ecotypes that collectively expand the range of light intensities over which the genus can thrive. In this paper, we explore this hypothesis by comparing the photophysiology of 10 different Prochlorococcus isolates from diverse oceanographic regimes. We found that the 10 isolates could be grouped into two loose clusters based on their growth response to varying light intensity and their chlorophyll b/a2 (Chl b/a2) ratios. Although both groups photoacclimate when grown over a range of light intensities, isolates with distinctly higher Chl b/a2 ratios (high B/A ecotype) reach maximal growth rates at lower irradiances (I(k,g)), have high growth efficiencies (α(g)), and are inhibited in growth at irradiances where isolates with low Chl b/a2 ratios (low B/A ecotype) are growing maximally. High Chl b/a2 ratios resulted in higher spectrally weighted average Chl a2-specific absorption coefficient (aβ(chl)/*), Chl a2-specific light-harvesting efficiency (α(chlα)), and quantum yield (φ(m)) under low growth irradiances for the isolates of the high B/A ecotype relative to the others. The distinction between the high and low B/A ecotypes is supported by molecular phylogenies constructed using the 16S ribosomal ribonucleic acid (rRNA) gene. The physiological differences between the ecotypes most likely result in different relative distributions in a given water column and in fluctuations in their relative abundances as a function of seasonal dynamics and water-column stability.