The affinity of Synechococcus (WH8102 and WH5701) alkaline phosphatases for different DOP model substrates was examined in laboratory experiments through its ability to inhibit the hydrolysis of the fluorogenic substrate 4-methylumbelliferyl phosphate (MUF-P). These data were collected as part of a study of "Dissolved organic phosphorus bond-class utilization by Synechococcus" (Waggoner et al., 2024). Study Abstract: Dissolved organic phosphorus (DOP) contains compounds with phosphoester (P-O-C), phosphoanhydride (P-O-P), and phosphorus-carbon (P-C) bonds. Despite DOP’s importance as a nutritional source for marine microorganisms, the bioavailability of each bond-class to the widespread cyanobacterium Synechococcus remains largely unknown. This study evaluates bond-class specific DOP utilization by cultures of an open ocean and a coastal ocean Synechococcus strain. Both strains exhibited comparable growth rates when provided phosphate, short-chain and long-chain polyphosphate (P-O-P), adenosine 5’-triphosphate (P-O-C and P-O-P), and glucose-6-phosphate (P-O-C) as the phosphorus source. However, growth rates on phosphomonoester adenosine 5’-monophosphate (P-O-C) and phosphodiester bis(4-methylumbelliferyl) phosphate (C-O-P-O-C) varied between strains, and neither strain grew on selected phosphonates. Consistent with the growth measurements, both strains preferentially hydrolyzed 3-polyphosphate, followed by adenosine 5’-triphosphate, and then adenosine 5’-monophosphate. The strains’ exoproteome contained phosphorus hydrolases, which combined with enhanced cell-free hydrolysis of 3-polyphosphate and adenosine 5’-triphosphate under phosphate deficiency, suggests active mineralization of short-chain polyphosphate by Synechococcus’ exoproteins. Synechococcus alkaline phosphatases presented broad substrate specificities, including activity towards short-chain polyphosphate, with varying affinities between the two strains. Collectively, these findings underscore the potentially significant role of compounds with phosphoanhydride bonds in Synechococcus phosphorus nutrition, thereby expanding our understanding of microbially-mediated DOP cycling in marine ecosystems.