Several attempts to separate hydrophobic tryptic and cyanogen bromide-digested short peptides from Na,K-ATPase, using HPLC and different aci—organic solvents gradients, failed because of the insolubility of the peptides in the initial or final solvents of the gradients used for elution. Therefore, we opted to use a detergent-containing mobile phase. For sodium dodecyl sulphate-solubilized tryptic peptides of M r 7·10 3–100·10 3, elution on a TSK-G3000SW size-exclusion column siccessfully separates families of peptides with a resolution of M r 5·10 3–10·10 3. Peptides in these size ranges can then be resolved completely by tricine—sodium dodecyl sulphate gel electrophoresis, and identified by microsequencing after transfer to polyvinylidene difluoride paper. For separation of smaller peptides a Biosep-SEC-S2000 column, eluted at slow flow-rates, was evaluated. Use of ammonium chloride buffer allows sensitive detection at 214 nm. The separated fractions are resolved and identified on 16.5% tricine gels. Reasonable resolution has been obtained with defined cyanogen bromide fragments of myoglobin. Resolution of small tryptic and cyanogen bromide fragments of Na,K-ATPase is less successful, but the experiments suggest ways of improving the resolution of peptides in the range M r 2·10 3–10·10 3.