Sphingosine kinase (two isoforms, SK1 and SK2) catalyse the conversion of sphingosine into sphingosine 1-phosphate and play a key role in health and disease. Each isoform has a predominant role in a disease-specific manner, such as in cancer, fibrosis and psoriasis. Therefore, it is important to develop isoform-specific inhibitors that can be translated to new medicines. Mapping of isoform amino acid sequence differences for SK2 onto the recently available crystal structures of SK1 suggests that subtle structural differences exist in the foot of the lipid-binding ‘J-channel’. Relatively conservative changes in compound structure can be used to change the activity profile of a ligand from ca. 100-fold SK1-selective inhibition, through equipotent SK1/SK2 inhibition, to reversed 100-fold SK2 selectivity, with retention of nanomolar potency. In addition, we have previously suggested that SK1 adopts a dimeric quaternary structure in which the alignment of contiguous hydrophobic and positively charged areas produces translocation from the cytoplasm and high-affinity attachment to the plasma membrane, required for activation of the kinase. A model involving release of a translocation brake and N-terminal/C-terminal inter-domain movement to enable translocation is proposed. These new aspects of the structure/function of SK1 provide novel avenues for drug development designed to block translocation and activation of SK1 in disease.