An enriched triad and terminal cisternae preparation was achieved from skeletal muscle through alterations of the differential centrifugation and muscle homogenization protocols. Both yield and specific activity (pmoles of radioligand binding per mg protein) were optimized for (3)H-PN200-110 (transverse tubule marker) and (3)H-ryanodine (terminal cisternae marker) binding sites. By pelleting crude microsomes between 2,000 an 12,000 x g without any rehomogenizations, we improved both the yield and specific activity of transverse tubule and terminal cisternae markers in crude microsomes by approximately 4-fold to 1000-3000 pmoles binding sites (starting material: approximately 400 grams wet weight fast twitch skeletal muscle), with 10-15 pmoles/mg. Rehomogenization of the 1,000 x g pellet, which is typically discarded, allowed recovery of an additional 5000 pmoles PN200-110 binding sites and an additional 8000 pmoles ryanodine binding sites. Crude microsomes from the rehomogenized 1,000 x g pellets typically displayed specific activities of 20-25 pmoles binding/mg for both (3)H-PN200-110 and (3)H-ryanodine. Separation of crude microsomes on a sucrose gradient increased specific activity up to a maximum of 50 pmoles/mg in a specific fraction, a five- to ten-fold increase over standard triadic or terminal cisternae preparations. The mean specific activity for enriched triads was 30-40 pmoles/mg for both PN200-110 and ryanodine in pooled fractions, while pooled fractions of enriched terminal cisternae displayed low (3)H-PN200-110 binding (3-5 pmoles/mg) and high (3)H-ryanodine-specific activity (30-40 pmoles/mg).