From fruit-bodies of the Japanese mushroom Boletus laetissimus two polyene pigments boletocrocin A and B were isolated and their structures determined by spectroscopic methods. The compounds represent diamides of hexadecaheptaenedioic acid with isoleucine and either aspartic acid or asparagine. The L-configuration of the amino acids was established after acid hydrolysis. The structures of five structurally related minor pigments were elucidated by LC-ESIMS.