The titin-cap protein telethonin was previously identified by our group as an interaction partner for the protein kinase D (PKD) catalytic domain, through a yeast two-hybrid screen of a human cardiac cDNA library. In the present work, kinase assays confirmed that recombinant full-length telethonin is a novel PKD substrate in vitro, and tandem mass spectrometric analysis using electron transfer dissociation identified S157 and S161 as putative PKD target sites. Further in vitro kinase assays using recombinant mutated telethonin in which S157 and S161 were replaced (either individually or in combination) by non-phosphorylatable Ala (S157A, S161A or S157/161A) confirmed that both S157 and S161, but no other sites, are targeted by PKD. A novel method for simultaneously detecting multiple phospho-moieties of telethonin, based on Phos-tag phosphate affinity SDS-PAGE, was developed and used to reveal that endogenous telethonin exists predominantly in a dually-phosphorylated form in isolated adult rat ventricular myocytes (ARVM) and in ventricular tissue from rat and mouse hearts. Experiments with heterologous expression by adenoviral gene transfer of epitope-tagged telethonin in wild type (WT) or mutated (S157/161A) form in ARVM indicated that WT telethonin becomes fully-phosphorylated, S157/161A telethonin is completely non-phosphorylatable, and the phosphorylation status of telethonin does not regulate its Z-disk localisation, as detected by immunolabelling and confocal microscopy. In a mouse model of pressure overload-induced left ventricular hypertrophy, significant but inverse changes were observed in myocardial telethonin expression (increased) and phosphorylation (decreased), suggesting stress-induced regulation of these processes and a potential link between telethonin protein turnover and its phosphorylation status. Further work is required to identify the cellular mechanisms that regulate telethonin phosphorylation and to determine the functional importance of such phosphorylation in physiological and pathological settings.