The bioavailability of the broccoli-derived sulphur-containing compounds, glucoraphanin (GR), S-methyl-L-cysteine sulfoxide (SMCSO) and sulforaphane (SF), were quantified through a dietary intervention study in humans with the use of soups made with three broccoli genotypes that differed in their Myb28 alleles. One genotype was homozygous for the normal broccoli Myb28B allele while the other two were either heterozygous or homozygous for a Myb28V allele introgressed from the wild species Brassica villosa. Phytochemical analysis of the soups confirmed that the presence of one or more Myb28V alleles led to higher levels of GR. A randomised, double-blinded, three-phase, crossover study was employed to quantify the pharmacokinetics of GR, SF and SMCSO following consumption of the soups. A significantly higher plasma concentration and urinary excretion of GR, SMCSO and SF and metabolites occurred following consumption of broccoli soup derived from the Myb28B/V and Myb28V/V genotypes compared to Myb28B/B genotype. There was considerable inter-individual variation in the excretion of these metabolites following consumption of the soups, with certain volunteers having consistently higher plasma levels of SF and higher levels of SF excretion for each of the different phases of the study. However, there was no correlation between plasma and urine levels of SF with SMCSO or GR. The use of in vitro models indicated that intact GR and SMCSO may either require a transporter to diffuse across the enterocytes or diffuse paracellularly to reach the systemic circulation. As an initial investigation to explore the biological activity of SMCSO, experiments were undertaken to investigate whether SMCSO could induce nrf2-regulated genes within human and mouse liver cells, with the use of SF as a positive control. No gene induction was observed with SMCSO and it was concluded that the biological activity of SMCSO was likely to reside in its microbial degradation products