Calcareous microplankton increased in abundance during the latest Jurassic, coinciding with the increase in abundance of calcareous nannofossils and with the onset of deposition of pelagic calcareous oozes. However, the timing and causes of the shift from microgranular tests of the earliest microplankton (chitinoidellids) to hyaline tests of calpionellids are obscured because the ultrastructure of two-layered praecalpionellids that occur during the Tithonian is poorly documented. Here, we investigate the ultrastructure of chitinoidellids and praecalpionellids from Upper Tithonian deposits in the Western Carpathians. We show that (1) the chitinoidellid microgranular layer is formed by elongated, euhedral, densely-packed, nanometric needles rather than by fragments of calcareous nannofossils, (2) two-layered chitinoidellids (Semichitinoidella) are formed by an internal microgranular layer (identical to that of Chitinoidella) and by an external hyaline prismatic layer, and (3) two-layered Praetintinnopsella exhibits an internal hyaline layer (with densely-packed, equant microcrystals) and an external layer formed by a dark organic rim. The external layer in Praetintinnopsella thus does not have any relation to the microgranular layer in chitinoidellids and the external hyaline layer of Semichitinoidella is not equivalent in structure to the hyaline layer of Praetintinnopsella. As both single-layer and two-layered chitinoidellids appear prior to the first appearance of Praetintinnopsella but still co-occur with this genus in the lowermost Upper Tithonian deposits, the origin of two-layered Praetintinnopsella either reflects a major transformation in biomineralization towards larger and more packed crystals during their earlier divergence from the chitinoidellid lineage or an origination of two-layered tests with a hyaline layer from an independent non-chitinoidellid ancestor.