•  Ca2+ release from the sarcoplasmic reticulum (SR) controls contraction in vertebrate skeletal muscle. Calsequestrin (CSQ) is thought to be the principal Ca2+ binding protein in the SR but little is known about SR Ca2+ content and loading characteristics, or CSQ isoform distribution, in human skeletal muscle fibres. •  Type I (slow‐twitch) and type II (fast‐twitch) skeletal muscle fibres in young healthy adults show highly‐stereotyped patterns of isoform expression of CSQ and SR Ca2+ pumps, in tight correspondence with isoform expression of the contractile proteins, which probably facilitates optimal contractile function in the individual fibre types. •  Endogenous Ca2+ content of the SR is slightly larger in type II fibres than in type I fibres, but its maximal capacity is substantially greater, probably due to the larger amount of the CSQ1 isoform present. SR Ca2+ content and capacity in type I fibres is probably determined by their content of both CSQ1 and CSQ2. Abstract  The relationship between sarcoplasmic reticulum (SR) Ca2+ content and calsequestrin (CSQ) isoforms was investigated in human skeletal muscle. A fibre‐lysing assay was used to quantify the endogenous Ca2+ content and maximal Ca2+ capacity of the SR in skinned segments of type I and type II fibres from vastus lateralis muscles of young healthy adults. Western blotting of individual fibres showed the great majority contained either all fast or all slow isoforms of myosin heavy chain (MHC), troponins C and I, tropomyosin and SERCA, and that the strontium sensitivity of the force response was closely indicative of the troponin C isoform present. The endogenous SR Ca2+ content was slightly lower in type I compared to type II fibres (0.76 ± 0.03 and 0.85 ± 0.02 mmol Ca2+ per litre of fibre, respectively), with virtually all of this Ca2+ evidently being in the SR, as it could be rapidly released with a caffeine‐low [Mg2+] solution (only 0.08 ± 0.01 and <0.07 mmol l−1, respectively, remaining). The maximal Ca2+ content that could be reached with SR Ca2+ loading was 1.45 ± 0.04 and 1.79 ± 0.03 mmol l−1 in type I and type II fibres, respectively (P < 0.05). In non‐lysed skinned fibres, where the SR remained functional, repeated cycles of caffeine‐induced Ca2+ release and subsequent Ca2+ reloading similarly indicated that (i) maximal SR Ca2+ content was lower in type I fibres than in type II fibres (P < 0.05), and (ii) the endogenous Ca2+ content represented a greater percentage of maximal content in type I fibres compared to type II fibres (∼59% and 41%, respectively, P < 0.05). Type II fibres were found on average to contain ∼3–fold more CSQ1 and ∼5–fold less CSQ2 than type I fibres (P < 0.001). The findings are consistent with the SR Ca2+ content characteristics in human type II fibres being primarily determined by the CSQ1 abundance, and in type I fibres by the combined amounts of both CSQ1 and CSQ2.