N,N'-Bis(carboxymethyl)-N,N'-dinitroso-1,4-phenylenediamine (1) fragments to release 1 equiv of NO center dot and the denitrosated radical of 1 (2), when exposed to a similar to 10 ns, 308 nm laser pulse. Species 2 can fragment to give another equivalent of NO center dot and the doubly denitrosated quinoimine derivative of 1 (3), it can recombine with NO center dot to give 1 and ring-nitrosated isomers of 1, or in the presence of a reducing agent, 2 can be reduced (to species 4). Photogenerated NO center dot can be used to probe fast reactions of biochemical interest, making 1 a valuable research tool. This paper focuses on the chemistry of 2, whose reactivity must be well characterized if 1 is to be used to its full potential. [Ru(NH3)(6)](2+) (Ru-II) and [Fe(CN)(6)](4-) (Fe-II) were both shown to reduce 2, with bimolecular rate constants in the diffusion limit. When solutions initially containing 70 mu M of Ru-II, 20 mu M myoglobin (Mb) and varying amounts of 1 were irradiated, the only Mb reaction product was nitrosomyoglobin (MbNO). In contrast, in solutions containing only Mb and 1, Mb is converted to both MbNO and oxidized myoglobin (metMb). When Fe-II was used in place of Ru-II, Mb was oxidized to metMb, but similar to 100x more slowly than in solutions containing only Mb and 1. This showed that 2 first oxidized Fe-II to [Fe(CN)(6)](3-) (Fe-III), which then oxidized Mb at the slower rate. The ratio metMb/MbNO obtained in the experiments with Fe-II was 0.6, whereas the ratio predicted from previously known chemistry of 2 was similar to 1 under the experimental conditions. The result is explained if, upon photolysis, 1 first forms a caged encounter complex [2, NO center dot], which fragments to give 3 and 2 equiv of NO center dot, without ever releasing free 2 into solution. This hypothesis was further strengthened by analyzing the amount of NO center dot generated by photolysis of 1 in the absence of added reductant. The original mechanism underestimates the NO center dot generated, a problem solved by invoking direct release of NO center dot and 3 from photolysis of 1.